Labeled peptides, proteins and antibodies and processes and intermediates useful for their preparation

ABSTRACT

The invention provides peptide synthons having protected functional groups for attachment of desired moieties (e.g. functional molecules or probes). Also provided are peptide conjugates prepared from such synthons, and synthon and conjugate preparation methods including procedures for identifying optimum probe attachment sites. Biosensors are provided having functional molecules that can locate and bind to specific biomolecules within living cells. Biosensors can detect chemical and physiological changes in those biomolecules as living cells are moving, metabolizing and reacting to its environment. Methods are included for detecting GTP activation of a Rho GTPase protein using polypeptide biosensors. When the biosensor binds GTP-activated Rho GTPase protein, an environmentally sensitive dye emits a signal of a different lifetime, intensity or wavelength than when not bound. New fluorophores whose fluorescence responds to environmental changes are also provided that have improved detection and attachment properties, and that can be used in living cells, or in vitro.

RELATED APPLICATIONS

This application is a continuation under 37 C.F.R. 1.53(b) of U.S.application Ser. No. 10/455,713 filed Jun. 3, 2003, which is acontinuation of International Application No. PCT/US20010/22194 filed onJul. 13, 2001, which claimed priority under 35 U.S.C. 119 of U.S.application Ser. No. 09/839,577 filed Apr. 20, 2001, U.S. ProvisionalApplication Ser. No. 60/279,302 filed Mar. 28, 2001, PCT/US2000/026821filed Sep. 29, 2000, and U.S. Provisional Application Ser. No.60/218,113 filed Jul. 13, 2000, which applications are incorporatedherein by reference.

GOVERNMENT FUNDING

The invention described herein was made with United States Governmentsupport under Grant Number MCB-9812248 awarded by the National ScienceFoundation, and under Grant Numbers CA58689, AG15430 and GM 57464awarded by the National Institutes of Health. The United StatesGovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

Modified peptides and proteins are valuable biophysical tools forstudying biological processes, both in vitro and in vivo. They are alsouseful in assays to identify new drugs and therapeutic agents. Inparticular, quantitative live cell imaging using fluorescent proteinsand peptides is revolutionizing the study of cell biology. An excitingrecent development within this field has been the construction ofpeptide and protein biosensors exhibiting altered fluorescenceproperties in response to changes in their environment, oligomericstate, conformation upon ligand binding, structure, or direct ligandbinding. Appropriately labeled fluorescent biomolecules allow spatialand temporal detection of biochemical reactions inside living cells. Seefor example Giuliano, K. A., et al., Annu. Rev. Biophys. Biomol. Struct.1995, 24:405-434; Day, R. N., Mol. Endocrinol. 1998, 12:1410-9; Adams,S. R., et al., Nature 1991, 349:694; Miyawaski, A., et al., Nature 1997,388:882-7; Hahn, K., et al., Nature 1992, 359:736; Hahn, K. M., et al.,J. Biol. Chem. 1990, 265:20335; and Richieri, G. V., et al., Mol. Cell.Biochem. 1999, 192:87-94.

Procedures for site-specific modification of polypeptides have beendescribed, including: chemically selective labeling in solution(Brinkley, M. Bioconjugate Chemistry 1992, 3:2-13) and on resin boundpeptides (Hackeng, T., et al., J. Biol. Chem. Submitted); introductionof ketone amino acids through synthetic procedures (Rose, K. J. Am.Chem. Soc. 1994, 116:30-33; King, T. P., et al., Biochemistry, 1986,25:5774-5779; Rose, K., et al., Bioconjugate Chem. 1996, 7:552-556;Marcaurelle, L. A., Bertozzi, C. R. Tett. Lett. 1998, 39:7279-7282; andWahl, F., Mutter, M. Tett. Lett. 1996, 37:6861-6864); and molecularbiology techniques (Cornish, V. W., et al., J. Am. Chem. Soc. 1996,118:8150). For example, green fluorescence protein (GFP) is afluorescent protein that has been fused to other proteins usingmolecular biology techniques and has been used to visualizeintracellular proteins (see, e.g., Katz et al., BioTechniques 25:298-304 (1998)).

While each of these methods has utility for producing a particular classof biosensor or labeled polypeptide, all have limitations that restricttheir general use. Labeling of natural amino acid side-chains insolution is often impractical because of the existence of many othercompeting nucleophiles. Additionally, the use of unnatural amino acids,such as those bearing ketones for selective labeling, requires thesynthesis of dye constructs or amino acids that are difficult to makeand are not available commercially. While a variety of proteins havebeen fused to GFP, some GFP-labeled proteins fail to fluoresce.Mutational analysis indicates that the structure of GFP is extremelysensitive to molecular or biochemical modifications. Moreover, GFP isextremely large (238 amino acids). Hence, fusion of GFP to otherproteins can alter the function of GFP or even the function of theprotein to which it is fused. Moreover, GFP does not have the diversecapabilities of smaller, synthetic molecules, some of which provide avariety of fluorescence wavelengths, or are capable of reporting onprotein conformation, or can photo-crosslink or act as NME or EPRprobes. Accordingly, new fluorescent molecules with more diverseproperties and new attachment methods are needed.

Currently, the major obstacles to the development of fluorescentbiosensors and labeled polypeptides remain: (1) The difficulty insite-specific placement of the dye in the polypeptide and (2)determining exactly which site is optimal for dye placement (Giuliano,K. A., et al., Annu. Rev. Biophys. Biomol. Struct. 1995, 24:405-434).Solvent-sensitive dyes and other biophysical probes must be placedprecisely for optimal response to changes in protein structure withoutinterference with biological activity. Also, the need for site-specificincorporation of two dyes without impairment of biological activity hasproven a serious limitation for utilization of fluorescence resonanceenergy transfer (FRET) within a single protein. Total chemical synthesisof proteins provides a potential solution to these problems (Wilken, J.,Kent, S. B. H. Curr. Op. Biotechnology. 1998, 9:412; Kent, S. B. H. Ann.Rev. Biochem. 1988, 57, 957-989; Dawson, P. E., et al., Science 1994,266:776-779; Muir, T. W., et al., Proc. Natl. Acad. Sci. 1998,95:6705-6710; and Cotton, G. J., et al., J. Am. Chem. Soc. 1999,121:1100-1101). However, many biophysical probes suitable forfluorescent biosensors or other purposes are not stable to the variousconditions used for peptide synthesis, and site-specific incorporationafter synthesis has been difficult to achieve.

Moreover, labeling with hydrophobic dyes such as thionine or methyleneblue can be problematic because these dyes autoaggregate in aqueoussolution at high concentration. See, J. Am. Chem. Soc. 63, 69 (1941).These aggregates cause a change in the absorption spectrum and areduction in the fluorescence of the dyes. Cyanines and merocyanines arealso thought to aggregate, causing a quenching of fluorescence (J. Phys.Chem. 69, 1894 (1965)). Such aggregation interferes with conjugation ofthese fluorescent dyes to other molecules such as proteins. Moreoveraggregation by cyanines and merocyanines can be exacerbated after thedyes are conjugated. For example, Waggoner et al. have observed anaggregation phenomenon following the conjugation of cyaninisothiocyanate with an antibody (Cytometry 10, 11-19 (1989)).Fluorescence of a conjugate between a cyanin fluorescent dye and ananti-HCG antibody (molar ratio=1.7) named CY5.18 is quenched incomparison with that of the free cyanin (see U.S. Pat. No. 5,268,486 andAnal. Biochem. 217, 197-204 (1994)). Also, while cyanines are generallystable, inexpensive, simple to conjugate to other molecules and of asuitable size for the recognition of small molecules, they do not changetheir fluorescence in response to environmental factors, such as solventpolarity. New dyes that eliminate these problems are needed.

Thus, there is currently a need for new fluorescent dyes and peptidesynthons having protected functional groups that can be selectivelymodified to incorporate one or more functional molecules (e.g. afluorescent label) following peptide synthesis. There is also a need forproteins and antibodies with biophysical probes attached to preciselocations, and for simple, non-destructive methods of making suchlabeled proteins and antibodies. Simpler methods for using these labeledpeptides, proteins and antibodies in vivo as biosensors are also needed.

SUMMARY OF THE INVENTION

The present invention provides a highly efficient method for thesite-specific attachment of biophysical probes or other molecules tounprotected peptides following chemical synthesis. The methodologyutilizes amino acids having one or more protected aminooxy groups, whichcan be incorporated during solid-phase peptide synthesis or which can becombined with recombinant peptides through post expression steps. It hasbeen discovered that the protected aminooxy group can be unmaskedfollowing peptide synthesis, and reacted with an electrophilic reagentto provide a modified (e.g. a labeled) peptide. The aminooxy groupreacts selectively with electrophiles (e.g. an activated carboxylicester such as an N-hydroxy-succinimide ester) in the presence of othernucleophilic groups including cysteine, lysine and amino groups.

Thus, selective peptide modification (e.g. labeling) can be accomplishedafter synthesis using commercially available and/or chemically sensitivemolecules (e.g. probes). The methodology is compatible with thesynthesis of C^(α)-thioester containing peptides and amide-formingligations, required steps for the synthesis of proteins by either totalchemical synthesis or expressed protein ligation. An aminooxy containingamino acid can be introduced into different sites by parallel peptidesynthesis to generate a polypeptide analogue family with each memberpossessing a single specifically-labeled site. The parallel synthesisenables the development of optimized biosensors or other modifiedpolypeptides through combinatorial screening of different attachmentsites for maximal response and minimal perturbation of desiredbiological activity.

Thus, a simple and efficient methodology for site-specific modification(e.g. labeling) of peptides after synthesis has been developed thatprovides high yield, selectivity, and compatibility with bothsolid-phase peptide synthesis and C^(α)-thioester peptide recombinantsynthesis.

Accordingly, the invention provides a synthetic intermediate (i.e. asynthon) useful for preparing modified peptides, which is a compound offormula (I):

wherein:

R¹ is hydrogen or an amino protecting group;

R² is hydrogen or a carboxy protecting group;

R is an organic radical comprising one or more aminooxy groups.

Peptides including one or more aminooxy groups are also useful syntheticintermediates that can be modified to provide related peptides havingaltered biological, chemical, or physical properties, such as, forexample, a peptide linked to a fluorescent label. Accordingly, theinvention also provides a peptide having one or more (e.g. 1, 2, 3, or4) aminooxy groups; provided the peptide is not glutathione. Theinvention also provides a peptide having one or more (e.g. 1, 2, 3, or4) secondary aminooxy groups.

The invention generally provides intermediates and methods that allowfor site-specific modification of peptides after synthesis. Accordingly,functional molecules can be selectively linked to a peptide to provide apeptide conjugate having altered biological, chemical, or physicalproperties. For example, functional molecules (e.g. biophysical probes,peptides, polynucleotides, and therapeutic agents) can be linked to apeptide to provide a peptide conjugate having differing and usefulproperties.

Thus, the invention also provides a compound of formula (III):

wherein:

R⁶ is a peptide, polypeptide or antibody;

X is a direct bond or a linking group;

R⁷ is hydrogen, (C₁-C₆)alkyl, an amino protecting group, or a radicalcomprising one or more aminooxy groups;

Y is a direct bond or a linking group; and

D is a functional molecule.

A functional molecule can be any label, dye, pharmaceutical, toxin,Preferably the functional molecule is a biophysical probe, such as afluorescent group that can be used for FRET studies or other studiesinvolving fluorescent signals, such as excimer pair formation.

Processes for preparing synthons of the invention as well as thepolypeptide, antibody and protein conjugates of the invention areprovided as further embodiments of the invention and are illustrated bythe procedures in the Examples below.

Thus, the invention also provides a method for preparing a peptideconjugate comprising a peptide and a functional molecule, comprisingreacting a peptide having one or more aminooxy groups with acorresponding functional molecule having an electrophilic moiety, toprovide the peptide conjugate.

The present invention further provides environment-sensing dyes that canbe readily conjugated to proteins and other molecules without theproblems of aggregation, fluorescence quenching and the like. Thepresent dyes strongly fluoresce and can fluoresce in anenvironmentally-sensitive manner suitable for use in living cells.Unlike cyanine dyes, the environmental sensitivity of these dyes can beused to form biosensors that can report many aspects of proteinbehavior, or the behavior of other molecules. Protein behaviorsincluding conformational change, phosphorylation state, ligandinteraction, protein-protein binding and various post-translationalmodifications affect the distribution of charged and hydrophobicresidues can be reported by the present dyes by changes in theirfluorescence.

The present invention overcomes the disadvantages of the availableenvironmentally-sensitive fluorescent dyes. The present fluorescentprobes exhibit high fluorescence levels before and after conjugation toother molecules, including peptides, proteins and antibodies, andchanges in fluorescence suitable for many purposes, including in vivoand in vitro assays of protein behavior.

The present invention provides new fluorescent dyes that can be used inany manner chosen by one of skill in the art. The dyes can be linked toany useful molecule known to one of skill in the art using any availableprocedure. In one embodiment the fluorescent dyes are linked topeptides, polypeptides or antibodies using the methods provided herein.These dyes have the following structure (IV).

wherein:

each m is separately an integer ranging from 1-3;

n is an integer ranging from 0 to 5;

R⁸, R¹¹ and R¹² are separately CO, SO₂, C═C(CN)₂, S, O or C(CH₃)₂;

each R¹³ is alkyl, branched alkyl or heterocyclic ring derivatized withcharged groups to enhance water solubility and enhance photostability;

R⁹ and R¹⁰ are chains carrying charged groups to enhance watersolubility (i.e. sulfonate, amide, ether) and/or chains bearing reactivegroups for conjugation to other molecules. The reactive group is afunctional group that is chemically reactive (or that can be madechemically reactive) with functional groups typically found inbiological materials, or functional groups that can be readily convertedto chemically reactive derivatives using methods well known in the art.In one embodiment of the invention, the charged and reactive groups areseparately haloacetamide (—NH—(C═O)—CH₂—X), where X is Cl, Br or I.Alternatively, the charged and reactive groups are separately amine,maleimide, isocyanato (—N═C═O), isothiocyanato(—N═C═S), acyl halide,succinimidyl ester, or sulfosuccinimidyl ester. In another embodiment,the charged and reactive groups are carboxylic acid (COOH), orderivatives of a carboxylic acid. An appropriate derivative of acarboxylic acid includes an alkali or alkaline earth metal salt ofcarboxylic acid. Alternatively, the charged and reactive groups arereactive derivatives of a carboxylic acid (—COORx), where the reactivegroup Rx is one that activates the carbonyl group of —COORx towardnucleophilic displacement. In particular, Rx is any group that activatesthe carbonyl towards nucleophilic displacement without beingincorporated into the final displacement product. Examples of COORx:ester of phenol or naphtol that is further substituted by at least onestrong electron withdrawing group, or carboxylic acid activated bycarbodiimide, or acyl chloride, or succinimidyl or sulfosuccinimidylester. Additional charged and reactive groups include, among others,sulfonyl halides, sulfonyl azides, alcohols, thiols, semicarbazides,hydrazines or hydroxylamines.

The invention still further provides a method of identifying an optimalposition for placement of a functional molecule on a peptide having apeptide backbone and a known activity, which includes making a series ofpeptide conjugates, each peptide conjugate having the same amino acidsequence and the same functional molecule, wherein the functionalmolecule is linked at a different location along the backbone of everypeptide conjugate in the series, and observing which functional moleculelocation does not substantially interfere with the known activity of thepeptide.

The invention also provides a method of identifying an optimal positionfor placement of a functional molecule in a protein having a knownactivity and an identified peptide segment for attachment of thefunctional molecule, which includes making a series of peptideconjugates, each peptide conjugate having the amino acid sequence of theidentified peptide segment and the same functional molecule, wherein thefunctional molecule is linked at a different location along the backboneof every peptide conjugate in the series; replacing the identifiedpeptide segment in each protein of a series of said proteins with apeptide conjugate selected from the series of peptide conjugates tocreate a series of protein conjugates each having the functionalmolecule at a different location; and observing which functionalmolecule location does not substantially interfere with the knownactivity of the protein.

The invention further provides a method of identifying an optimalposition for placement of an environmentally-sensitive functionalmolecule on a peptide biosensor having a backbone, which includes makinga series of peptide conjugates, each peptide conjugate having the sameamino acid sequence and the same functional molecule, wherein thefunctional molecule is at a different location along the backbone ofevery peptide conjugate in the series, and observing which functionalmolecule location provides the strongest signal change in response to anenvironmental change in the peptide conjugate. The signal change can beany observable change in a signal, for example, the change can be achange in fluorescence emission intensity, fluorescence duration orfluorescence emission wavelength. The environmental change in thepeptide biosensor can be, for example, an interaction with a targetmolecule.

The invention still further provides a method of identifying an optimalposition for placement of an environmentally-sensitive functionalmolecule in a protein having a known activity and an identified peptidesegment for attachment of the functional molecule, which includes makinga series of peptide conjugates, each peptide conjugate having the aminoacid sequence of the identified peptide segment and the sameenvironmentally-sensitive functional molecule, wherein theenvironmentally-sensitive functional molecule is linked at a differentlocation along the backbone of every peptide conjugate in the series;replacing the identified peptide segment in each protein of a series ofsaid proteins with a peptide conjugate selected from the series ofpeptide conjugates to create a series of protein conjugates, each havingthe environmentally-sensitive functional molecule at a differentlocation; and observing which functional molecule location provides thestrongest signal change in response to an environmental change in theprotein conjugate.

The invention also provides a method for detecting GTP activation of aRho GTPase protein, which includes contacting a polypeptide biosensorwith a test substance, wherein said polypeptide biosensor comprises apolypeptide capable of binding a GTP-activated Rho GTPase protein, andwherein said polypeptide is operatively linked to an environmentallysensitive fluorescent dye; and observing fluorescence emissions from thepolypeptide biosensor at a wavelength emitted by said fluorescentacceptor dye; wherein the environmentally sensitive fluorescent dye willemit light of a different intensity or a different wavelength when thepolypeptide biosensor is bound to the GTP-activated Rho GTPase proteinthan when the polypeptide biosensor is not bound.

The invention further provides a method of detecting the location of acellular protein within a living cell that includes providing the livingcell with a biosensor capable of binding to a tag on the cellular targetprotein; and detecting the location of a functional molecule on thebiosensor within the living cell. The tag on the cellular target proteincan be a peptide segment that has been fused to the cellular protein. Inone embodiment, the tag is a peptide which includes SEQ ID NO:16 andthat can bind to a biosensor having a peptide segment with SEQ ID NO:15.The biosensor can include a peptide-conjugate of the invention. Any ofthe functional molecules, pharmaceuticals, toxins, labels, dyes orcompounds can also be present on the biosensor. Moreover, any cellularprotein can be detected using this method, for example, calmodulin, RhoGTPase, rac, cdc42, mitogen-activated protein kinase, Erk1, Erk2, Erk3,Erk4, IgE receptor (F_(c)εRI), actin, α-actinin, myosin, or a majorhistocompatibility protein. The methods provided herein can detect andidentify the cellular location of proteins and cellular proteins thathave never been successful labeled and observed in vivo.

BRIEF DESCRIPTION OF THE FIGURES

In the following detailed description of example embodiments of theinvention, reference is made to the accompanying drawings which form apart hereof, and which is shown by way of illustration only, specificembodiments in which the invention may be practiced. It is to beunderstood that other embodiments may be utilized and structural changesmay be made without departing from the scope of the present invention.

FIG. 1 illustrates a general strategy for site-specific labeling ofpolypeptides. The protected aminooxy group is incorporated duringsolid-phase peptide synthesis (synthesis on a thioester-linker resin isshown); cleavage from the resin generates a peptide possessingunprotected sidechains, an aminooxy group and a C-terminal thioester;and ligation and subsequent site-specific labeling produces thefull-length peptide with a functional molecule attached at the aminooxynitrogen.

FIG. 2 illustrates the synthesis of PA-test and SA-test peptides.

FIG. 3 shows HPLC analysis of purified SA-test peptide (top) and crudereaction products from optimized labeling conditions (bottom).

FIG. 4 illustrates the synthesis of a protected intermediate (4) of theinvention.

FIG. 5 illustrates one method for using biosensors according to thepresent invention. In this example, the activation of Rac1 (“RAC”) byGTP was observed. A fragment of p21-Activated kinase (PAK) capable ofbinding to Rac1 (termed “PBD”) was used as a biosensor because PAK willonly bind to Rac1 when Rac1 is activated by GTP. A Rac1-GreenFluorescent Protein fusion protein (shown as a square named “RAC”attached to a circle named “GFP”) was made and a cell line expressingthis fusion protein was generated. Cells expressing RAC-GFP wereinjected with PBD labeled with Alexa-546 dye (“Dye”). The PBD biosensorbinds selectively to GTP-RAC-GFP, but not to GDP-RAC-GFP. Upon bindingto GTP-RAC-GFP, the Alexa on the labeled fragment undergoes fluorescenceresonance energy transfer (FRET) as the Alexa and GFP fluorophores arebrought close together. This FRET can be measured within a living cellor in vitro to map the distribution, localization and level of Rac-GTPactivation. FRET produces a fluorescence signal which is distinct from aGFP fluorescence signal because energy is transferred from the excitedGFP fluorophore to the nearby Alexa dye (J. R. Lakowicz, Principles ofFluorescence Spectroscopy (Plenum Press, New York, 1983), pp. 305-341)).By imaging the cell with different wavelengths, both the distribution ofRac and Rac activation can be studied in the same cell. GFP excitationand emission are used for overall Rac distribution, while GFP excitationand Alexa emission are used for FRET.

FIG. 6 illustrates one method of using an environmentally sensitivefluorescent dye in the present methods so that changes in naturallyexisting proteins can be detected and observed in vivo or in vitro.

FIG. 6 a depicts FRET between the Rac1-Green Fluorescent Protein(GFP-Rac1) fusion and the p21-activated kinase biosensor (PBD) labeledwith Alexa-546 dye as shown in FIG. 5. Inactive Rac1 is depicted as alarger gray circle with Green Fluorescent Protein (smaller circle)attached. Upon activation by GTP, Rac1 undergoes a structural changedepicted as a gray circle changing to a half-rounded gray rectangle.Unbound PBD is depicted as a black L-shape with an attached Alexa-546dye (open circle). Before Rac1 is activated, PBD cannot bind and theAlexa-546 cannot undergo FRET. However, after Rac1 activation, the Rac1assumes a conformation that permits PBD binding. Such binding juxtaposesthe Green Fluorescence Protein and the Alexa-546, which produces FRET.

FIG. 6 b illustrates how an environmentally sensitive fluorescent dyeeliminates the need to create a fusion protein like the GFP-Rac1 proteindepicted in FIG. 6 a. In FIG. 6 b, a natural, unmodified protein isdepicted as a gray oval. The protein changes conformation uponactivation by GTP, depicted as the transition to a half-rounded grayrectangle. When the protein is in the activated state, apolypeptide-biosensor that binds only to the activated state (blackL-shape), with an attached environmentally sensitive dye (open circle),can bind. Upon binding, the environmentally sensitive dye will emitlight of a different wavelength, duration or intensity (filled circle)than before binding. Use of this type of environmentally sensitive dyeis further illustrated in the Figures to follow.

FIG. 7 illustrates what conditions will optimally provide FRET betweenGFP-Rac and Alexa-PBD in vitro. FIG. 7A shows fluorescence emission fromsolutions containing a fixed level of GFP-Rac bound to GTPγS atdifferent concentrations of Alexa-PBD (PBD labeled with Alexa-546).Light at 480 nm was selectively used for GFP excitation, and direct(non-FRET) excitation of Alexa was subtracted from these spectra. In theabsence of Alexa-PBD, the emission from GFP (peak at 508 nm) is maximaland no Alexa emission (peak at 568 nm) is seen. As the concentration ofAlexa-PBD is increased, binding of Alexa-PBD to Rac-GFP leads to FRET,producing increasing emission at 568 nm and a decrease at 508 nm. FIG.7B shows the variation of the 568/508 nm emission ratios with changes inthe level of GTP (solid circles) or GDP (solid squares). All data pointswere the average of three independent experiments.

FIG. 8 illustrates how GFP-Rac expression levels and levels ofintracellular Alexa-PBD (as observed by fluorescence) correlate withchanges in normal cell behavior produced by these proteins. FIG. 8Ashows what levels of GFP-Rac expression, as measured by log GFPintensity per cell area, were correlated with ruffling. Cells withdifferent expression levels of either wild type or a constitutivelyactive Q6 IL mutant of GFP-Rac were scored for ruffling. Each pointrepresents an individual cell, placed in the higher (Ruffling) or lower(Nonruffling) row depending on whether ruffling was induced. Asillustrated, there is a level of GFP intensity below which ruffling wasconsistently not induced by expression of wild type GFP-Rac. Only cellswith Rac expression levels below 250 on this scale were used inbiological experiments. The validity of this approach was supported byscoring of constitutively active Rac, which showed ruffle induction atmuch lower levels of expression.

FIG. 8B shows which levels of intracellular Alexa-PBD would perturbnormal serum-induced ruffling, as observed by Alexa intensity per cellarea. Cells were scored as in FIG. 8A, at different levels of Alexa-PBDfluorescence. Based on this experiment, only cells with Alexa-PBDconcentrations below 400 intensity units on this scale were used inbiological experiments. These studies demonstrated that FRET could stillbe readily detected at appropriately low levels of introduced protein.

FIG. 9 illustrates the dynamics of Rac activation during growth factorstimulation of quiescent cells. FIG. 9A provides photomicrographsshowing Rac localization (GFP-Rac) and Rac activation (FRET) beforestimulation of quiescent Swiss 3T3 fibroblasts. FIG. 9B providesphotomicrographs of the same Swiss 3T3 fibroblasts three minutes afteraddition of serum. Wanner or lighter colors correspond to higherintensity values. The cells showed accumulation of Rac at and around thenucleus before stimulation (GFP-Rac image). Most of the nuclear GFP-Racwas associated with the nuclear envelope. Serum or PDGF additiongenerated multiple moving ruffles that showed FRET, while no FRET wasseen at the nucleus before or after stimulation. Of thirty-five cellsstimulated with either serum or PDGF, thirty-one began ruffling within15 minutes. FRET was seen in the ruffles of all but one of the rufflingcells.

FIGS. 9C and 9D demonstrate that simple localization of Alexa-PBD isinferior to FRET in quantifying and localizing Rac-GTP binding (Bar=8μm). The ruffle in FIG. 9B is shown in close-up in FIG. 9C, visualizedusing FRET. PBD localization in the same region is visualized usingAlexa fluorescence in FIG. 9D, with scaling optimized for detection ofthe ruffle. Without prior knowledge of the ruffle's location, thislocalization would have been difficult to discern. The high backgrounddue to unbound PBD cannot be eliminated in FIG. 9D and binding to othertarget proteins is not eliminated as it is in the highly specific FRETsignal.

In each of the GFP-Rac images above, intensities range between 300-1100.The image of FRET before serum addition was scaled to demonstrate thelow levels of FRET, with values ranging between 0 and 15. In the imageof FRET after stimulation, the ruffle contains the highest values of 40to 65. Nuclear FRET was not seen in any of the cells examined.

FIG. 10 illustrates Rac activation in motile Swiss 3T3 fibroblast cells.This figure shows two examples of where a Rac 1 activation gradient isformed, in confluent monolayer cells and in “wound healing” cells. HighRac1 activation occurred at the leading edge of motility, particularlyin wound healing cells. In these experiments high levels of Rac-GTP werefrequently seen in the juxtanuclear region of the cell (FIG. 10A). Thestrong correlation of this gradient with the direction of movementindicates that activated Rac is spatially organized in polarized cellsto help guide or propagate movement. Comparison of the GFP (FIG. 10A)and FRET (FIG. 10B) images shows that the distribution of activated Racdoes not parallel that of Rac 1 itself. FRET intensities (FIG. 10B) are0-18 (top image) and 0-32 (bottom image). In the GFP images (FIG. 10A),intensities range between 98-700 (top image) and 100-1100 (bottomimage).

FIG. 11 provides the structure of one of the present fluorescent dyesand the spectrum of fluorescence emission for that dye in water,methanol and butanol. As illustrated, the fluorescent emission of thisdye increases with increasing solvent hydrophobicity. This figureillustrates the environmental-sensitivity of this dye.

FIG. 12 provides the structure of another fluorescent dye of the presentinvention and shows its spectrum in aqueous solution, compared tosimilar dyes lacking the groups designed to prevent aggregation. In thecurve from each dye, the peak to the right is the unconjugated, highlyfluorescent form of the dye, while that to the left is the weaklyfluorescent form. The curve furthest to the right is the dye containinggroups to prevent aggregation. As illustrated, the chosen groups helpprevent aggregation of the dye.

FIG. 13 provides one method for synthesizing a dye of the presentinvention. Conversion of compound 1 to an amine 2 followed by protectionof the amine provides compound 3, which can be alkylated to givecompound 4. Reaction of compound 4 with compound 9 provides compound 5,which can be deprotected to provide amine 6. Alkylation or acylation ofamine 6 with a chain carrying a charged group, or with a chain bearing areactive group for conjugation to another molecule, or with anothermolecule directly provides a dye of the invention 7. Intermediatecompound 9 can be prepared by condensation of compound 8 with therequisite aldehyde, under conditions that are known in the art

FIG. 14 provides a three-dimensional image of the CRIB domain (“CBD”) ofthe Wiscott-Aldrich syndrome protein (WASP) bound to Cdc42. Theessential residues are depicted in yellow, hydrophobic residues depictedin purple and the red sites show positions where the new dyes wereattached to generate changes in fluorescence when the CBD bound Cdc42.

FIG. 15 provides the intensity of fluorescence at various wavelengthsobserved when fluorescently labeled CBD binds to cdc42. When cdc42 isactivated with GTPγS, highly intense fluorescence is observed (linelabeled “GTPγS”), compared to when no cdc42 is present (line labeled “nocdc42”), or when cdc42 is not activated (line labeled “GDP”).

FIG. 16 shows how the present methods can be used for in vitro assays oncrude cellular lysates. In this case, the fluorescently labeled CBD wasadded to a neutrophil lysate. Upon binding to activated cdc42, CBD willemit fluorescence of greater intensity. At time zero fMLP, whichstimulates neutrophils to activate cdc42, was added to the cellularlysate and the amount of fluorescence generated by the lysate (●) wasmeasured as a function of time. As a control, the maximal amount offluorescence that could be obtained from the lysate was estimated byadding saturating levels of GTPγS (▾), which would activate most or allof the cdc42 in the cellular lysate. In this manner the levels of cdc42in a cellular population can be quantified.

FIG. 17 provides photomicrographs of live cells injected withfluorescently labeled CBD. The lighter colors indicate where theactivated cdc42 is present within the cells. The merocyanin dye (“mero”)is an environmentally sensitive dye that will fluoresce at higherintensity upon binding of the CBD-mero conjugate to activated cdc42. Inthis case, the intensity of fluorescence emitted by anon-environmentally sensitive fluorophore (Alexa) linked to CBD wasdetermined relative to the intensity of fluorescence emitted by theCBD-mero fluorophore. The ratio of the two permitted backgroundfluorescence intensity to be subtracted so that the fluorescence fromactivated cdc-42 could be localized with greater precision.

FIG. 18 illustrates the affinity of the labeled leucine zipper peptidebiosensor for the leucine peptide tag as determined by equilibriumfluorescence titration, which monitored the changes in anisotropy (o)and fluorescence intensity (●) as the amount of tag binding peptide wasincreased. Upon binding, the labeled biosensor showed a drop in quantumyield and a more than 230% increase in rhodamine anisotropy, indicatingconsiderable reduction in rotation of the dye. The anisotropy valuesplotted against the unlabeled peptide tag concentration were fit to anequation describing this interaction as a function of unlabeled peptideconcentration, indicating a tight interaction with a dissociationconstant (Kd) of 5.4±1.1 nM.

FIG. 19 provides photomicrographs of the same cell with visualization ofα-actinin by fluorescently labeled GFP-α-actinin and a rhodamine peptidebiosensor of the invention. Cos-7 cells were transfected with anα-actinin construct with GFP fused to the N-terminus and the tag peptideon the C-terminus. The tag peptide and the rhodamine-peptide biosensorbound as illustrated in FIG. 18. The rhodamine-peptide biosensor wasinjected into these cells and α-actinin localization was visualizedusing GFP or rhodamine fluorescence. FIGS. 19 a and 19 c show GFP imagesand FIGS. 19 b and 19 d show rhodamine images taken of the same cells.The similar fluorescence distribution in the GFP image (FIG. 19 a) andrhodamine image (FIG. 19 b) demonstrate labeling of α-actinin with ahigh degree of specificity. FIGS. 19 c (GFP) and 19 d (rhodamine) showdeconvolution imaging of the same cell. This technique removesout-of-focus light, to demonstrate more clearly the coincidence ofemission from the two fluorophores. (Bar=10 microns).

FIG. 20 provides photomicrographs of a different cell than is shown inFIG. 19, with visualization of α-actinin by fluorescently labeledGFP-α-actinin and a rhodamine peptide biosensor of the invention asdescribed in FIG. 19. FIG. 20 a provides GFP fluorescence and FIG. 20 bprovides rhodamine fluorescence. (Bar=10 microns).

FIG. 21 is a representative example from control experiments in whichcells were transfected with the GFP construct described in FIG. 18, butnot injected with rhodamine peptide. FIG. 21 a shows GFP fluorescenceand FIG. 21 b shows rhodamine fluorescence, taken under exposureconditions and with fluorescence levels similar to those in FIGS. 19 and20. This control demonstrated that the rhodamine fluorescence was notsimply ‘bleedthrough’ of GFP into the rhodamine image. Similar resultswere obtained by injecting rhodamine peptide biosensor intonontransfected cells, showing that the GFP fluorescence was not due torhodamine ‘bleedthrough’ (data not shown). (Bar=10 microns).

FIG. 22 provides photomicrographs illustrating fluorescent labeling ofan endoplasmic reticulum membrane protein in vivo. A subunit of the Fereceptor (F_(c)εRI α), a protein which spans the endoplasmic reticulummembrane, was tagged with. Cells were cotransfected with an MHC-GFPfusion protein as an endoplasmic reticulum marker, and Fe receptor fusedto the peptide tag. The cells were then injected with therhodamine-labeled peptide and imaged in both rhodamine and GFP channels.FIG. 22 a shows the GFP fluorescence of the MHC marker, FIG. 22 c showsthe rhodamine fluorescence of the tagged F_(c)εRIα receptor. The imagesfrom FIGS. 22 a and c are merged in FIG. 22 b. The MHC-GFP is morebroadly distributed relative to the Fe receptor, which appears toconcentrate in ER regions closer to the nucleus and in the nuclearmembrane. Note that the GFP-expressing cell which was not injected withrhodamine does not artefactually appear in the rhodamine image,indicating again that similar rhodamine and GFP fluorescence are not dueto ‘bleedthrough’ of GFP fluorescence into the rhodamine image (Bar=10microns).

FIG. 23 shows the fluorescence of the ERK2-dye construct under differentconditions. ERK2 was activated with MEK for 0-60 min, as indicated inthe figure. When Mg and ATP were added, ERK2 was phosphorylated; no suchphosphorylation was observed when no Mg, ATP or MEK was present. Thedata shown here demonstrate that MEK can interact with the labeledprotein.

FIG. 24 shows the fluorescence intensity as a function of wavelength forERK2 when ERK2 is phosphorylated and unphosphorylated. Approximately1.6-fold increase in emission was observed, when the Erk2 wasphosphorylated.

FIG. 25 shows the fluorescence of ERK2, in the presence and absence ofMEK, as a function of time of incubation with MEK. No substantial Erk2fluorescence was observed when MEK was not present. This resultdemonstrated the suitability of the present methods for detectingactivation of ERK2 in live cells.

DETAILED DESCRIPTION

The following definitions are used, unless otherwise described.

Alkylene, alkenylene, alkynylene, etc. denote both straight and branchedgroups; but reference to an individual radical such as “propylene”embraces only the straight chain radical; a branched chain isomer suchas “isopropylene” being specifically referred to. Aryl denotes a phenylradical or an ortho-fused bicyclic carbocyclic radical having about nineto ten ring atoms in which at least one ring is aromatic.

The term “amino acid,” includes the residues of the natural amino acids(e.g. Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Hyl, Hyp, Ile, Leu,Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) in D or L form, as wellas unnatural amino acids (e.g. phosphoserine, phosphothreonine,phosphotyrosine, hydroxyproline, gamma-carboxyglutamate; hippuric acid,octahydroindole-2-carboxylic acid, statine,1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, penicillamine,omithine, citruline, α-methylalanine, para-benzoylphenylalanine,phenylglycine, propargylglycine, sarcosine, and tert-butylglycine). Theterm also includes natural and unnatural amino acids bearing aconventional amino protecting group (e.g. acetyl or benzyloxycarbonyl),as well as natural and unnatural amino acids protected at the carboxyterminus (e.g. as a (C₁-C₆)alkyl, phenyl or benzyl ester or amide).Other suitable amino and carboxy protecting groups are known to thoseskilled in the art (See for example, T. W. Greene, Protecting Groups InOrganic Synthesis; Wiley: New York, 1981, and references cited therein).

The term “peptide” includes any sequence of 2 or more amino acids. Thesequence may be linear or cyclic. For example, a cyclic peptide can beprepared or may result from the formation of disulfide bridges betweentwo cysteine residues in a sequence. Thus, the term includes proteins,enzymes, antibodies, oligopeptides, and polypeptides. Peptide sequencesspecifically recited herein are written with the amino terminus on theleft and the carboxy terminus on the right.

An “aminooxy group” is a group having the following formula

wherein the open valences are filled by any acceptable radical. A“secondary aminooxy group” is an aminooxy group where one of the openvalences on the nitrogen is filled by a radical other than a hydrogen.

The term “functional molecule” includes any compound that can be linkedto a peptide to provide a peptide conjugate having useful properties.Such conjugates may be useful for studying the structure or function ofthe peptide, or a polypeptide, antibody, antigen or other protein. Suchconjugates can also be used for therapeutic treatments and diagnoses.Functional molecules linked to peptides by the present methods can beused in any assay, procedure or tracing protocol known to one of skillin the art. The functional molecules may also be used with biosensors ascontemplated below. Peptide-conjugates with such functional moleculesmay be useful for drug screening, as pharmacological tools, as researchtools, or as therapeutic agents. For example, the term functionalmolecule includes labels, dyes, ESR probes, reporting groups,biophysical probes, peptides, polynucleotides, therapeutic agents,pharmaceuticals, toxins, cross-linking groups (chemical orphotochemical), a compound that modifies the biological activity of thepeptide, or a caged molecule (e.g. a reporting molecule or abiologically active agent that is masked and that can be unmasked byphotoactivation or chemical means).

The term “biophysical probe” includes any group that can be detected invitro or in vivo, such as, for example, a fluorescent group, aphosphorescent group, a nucleic acid indicator, an ESR probe, anotherreporting group, a moiety, or a dye that is sensitive to pH change,ligand binding, or other environmental aspects.

Amino acids and peptides that include one or more aminooxy groups areuseful intermediates for preparing peptide conjugates. The aminooxygroup(s) can typically be positioned at any suitable position on theamino acid or peptide. For example, the aminooxy group(s) canconveniently be incorporated into the side chain of the amino acid orinto one or more side chains of the peptide. Thus, as used herein withrespect to the amino acids and peptides of the invention, the term “aradical comprising one or more aminooxy groups” includes any organicgroup that can be attached to the amino acid or peptide that includesone or more aminooxy groups. For example, the term includes a carbonchain having two to ten carbon atoms; which is optionally partiallyunsaturated (i.e. contains one or more double or triple bonds); whichchain is optionally interrupted by one or more (e.g. 1, 2, or 3) —NH—,—O—, or —S—; which chain is optionally substituted on carbon with one ormore (e.g. 1, 2, or 3) oxo (═O) groups; and which chain is optionallysubstituted with one or more (e.g. 1, 2, or 3) aminooxy groups.Preferably, the aminooxy group(s) are secondary aminooxy groups.

The term “cross-linking group” refers to any functionality that can forma bond with another functionality, such as photoaffinity label or achemical crosslinking agent.

The term “caged molecule” includes a molecule or reporter group that ismasked such that it can be activated (i.e. unmasked) at a given time orlocation of choice, for example using light or a chemical agent.

Specific and preferred values listed below for radicals, substituents,and ranges, are for illustration only; they do not exclude other definedvalues or other values within defined ranges for the radicals andsubstituents.

Specifically, (C₁-C₆)alkylene can be methylene, ethylene, propylene,isopropylene, butylene, iso-butylene, sec-butylene, pentylene, orhexylene; (C₃-C₈)cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, or cyclooctyl; (C₂-C₆)alkenylene can bevinylene, allylene, 1-propenylene, 2-propenylene, 1-butenylene,2-butenylene, 3-butenylene, 1,-pentenylene, 2-pentenylene,3-pentenylene, 4-pentenylene, 1-hexenylene, 2-hexenylene, 3-hexenylene,4-hexenylene, or 5-hexenylene; (C₂-C₆)alkynylene can be ethynylene,1-propynylene, 2-propynylene, 1-butynylene, 2-butynylene, 3-butynylene,1-pentynylene, 2-pentynylene, 3-pentynylene, 4-pentynylene,1-hexynylene, 2-hexynylene, 3-hexynylene, 4-hexynylene, or 5-hexynylene;and aryl can be phenyl, indenyl, or naphthyl;

A specific value for R is a radical of formula (V):

wherein

R³ is hydrogen, (C₁-C₆)alkyl, an amino protecting group, or a radicalcomprising one or more aminooxy groups;

R⁴ is hydrogen, or an amino protecting group; and

R⁵ is hydrogen, or (C₁-C₆)alkyl.

A specific value for R¹ is hydrogen or benzyloxycarbonyl.

A specific value for R² is hydrogen.

A specific value for R³ is methyl.

A specific value for R⁴ is hydrogen, 2-chlorobenzyloxycarbonyl, orbenzyloxycarbonyl.

A specific value for R⁵ is hydrogen.

A specific value for R⁶ is an antibody.

A specific value for R⁶ is a peptide or polypeptide or antibody thatincludes about 2 to about 1000 amino acids. A more specific value for R⁶is a peptide that includes about 5 to about 500 amino acids. An evenmore specific value for R⁶ is a peptide that includes about 10 to about100 amino acids.

Specifically X is a linking group that is about 5 angstroms to about 100angstroms in length. More specifically, X is a linking group of about 5angstroms to about 25 angstroms in length.

Specifically X is —R_(a)—C(═O)—NH—R_(b)— wherein each of R_(a) and R_(b)is independently (C₁-C₆)alkylene. Preferably, each of R_(a) and R_(b) ismethylene (—CH₂—).

A preferred value for R⁶ is KKKEKERPEISLPSDFEHTIHVGFDACTGEFTGMPEQWARLLQT (SEQ ID NO: 1) or an antibody.

A specific value for R⁷ is hydrogen.

Another specific value for R⁷ is (C₁-C₆)alkyl.

A preferred value for R⁷ is methyl.

Specifically Y is a linking group that is about 5 angstroms to about 100angstroms in length. More specifically, Y is a linking group of about 5angstroms to about 25 angstroms in length.

A specific value for Y is (C₁-C₆)alkylene.

A preferred value for Y is methylene (—CH₂—).

Fluorescent Dyes

Any fluorescent dye known to one of skill in the art is contemplated bythe present invention as a functional molecule. A fluorescent dye can beexcited to fluoresce by exposure to a certain wavelength of light. Whenused as the reporter molecule of the biosensors of the presentinvention, the dye is preferably environmentally sensitive. As usedherein, “environmentally sensitive” means that the signal from thefunctional molecule changes when the peptide, polypeptide or antibodyinteracts with, or becomes exposed to, a different environment. Forexample, when the environmentally sensitive functional molecule is afluorescent dye, the fluorescence from that fluorescent dye will changeas the environment changes. In one embodiment an environmentallysensitive fluorescent dye attached to a peptide, polypeptide or antibodywill fluoresce differently upon target binding by the peptide,polypeptide or antibody to which the dye is attached. Any dye whichemits fluorescence and whose fluorescence changes when the pH or thehydrophilicity/hydrophobicity of the environment changes is anenvironmentally sensitive dye contemplated by the present invention.

Preferred fluorescent groups include molecules that are capable ofabsorbing radiation at one wavelength and emitting radiation at a longerwavelength, such as, for example, Alexa-532, Hydroxycoumarin,Aminocoumarin, Methoxycoumarin, Coumarin, Cascade Blue, Lucifer Yellow,P-Phycoerythrin, R-Phycoerythrin, (PE), PE-Cy5 conjugates, PE-Cy7conjugates, Red 613, Fluorescein, BODIPY-FL, BODIPY TR, BODIPY TMR, Cy3,TRITC, X-Rhodamine, Lissamine Rhodamine B, PerCP, Texas Red, Cy5, Cy7,Allophycocyanin (APC), TruRed, APC-Cy7 conjugates, Oregon Green,Tetramethylrhodamine, Dansyl, Dansyl aziridine, Indo-1, Fura-2, FM 1-43,DilC18(3), Carboxy-SNARF-1, NBD, Indo-1, Fluo-3, DCFH, DHR, SNARF,Monochlorobimane, Calcein, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amine(NBD), ananilinonapthalene, deproxyl, phthalamide, amino pH phthalamide,dimethylamino-naphthalenesulfonamide, probes comparable to Prodan,Lordan or Acrylodan and derivatives thereof. Coumarin fluorescent dyesinclude, for example, amino methylcoumarin,7-diethylamino-3-(4′-(1-maleimidyl)phenyl)-4-methylcoumarin (CPM) andN-(2-(1-maleimidyl)ethyl)7-diethylaminocoumarin-3-carboxamide (MDCC).Preferred fluorescent probes are sensitive to the polarity of the localenvironment and are available to those of skill in the art.

Other useful functional molecules include those that displayfluorescence resonance energy transfer (FRET). Many such donor-acceptorpairs are known, and include fluorescein to rhodamine, coumarin tofluorescein or rhodamine, etc. Still another class of useful label pairsinclude fluorophore-quencher pairs in which the second group is aquencher, which decreases the fluorescence intensity of the fluorescentgroup. Some known quenchers include acrylamide groups, heavy atoms suchas iodide and bromate, nitroxide spin labels such as TEMPO, etc. Thesecan be adapted for use as environmentally sensitive functional moleculesof biosensors.

Exemplary fluorescent proteins which can be used to label the presentpeptides, polypeptides and antibodies include green fluorescent protein(GFP), cyan fluorescent protein (CFP), red fluorescent protein (RFP),yellow fluorescent protein (YFF), enhanced GFP (EGFF), enhanced YFP(EYFP), and the like.

New Fluorescent Dyes

The present invention also provides novel fluorescent dyes that retainhigh fluorescence emission after conjugation to other molecules andavoid problems of aggregation and insolubility. These dyes areparticularly preferred for many of the imaging methods and conjugatescontemplated but need not be restricted to use in the methods andconjugates contemplated herein. Thus, the present invention is directedto highly fluorescent dyes of structure IV wherein, each m is separatelyan integer ranging from 1-3;

n is an integer ranging from 0 to 5;

R⁸, R¹¹ and R¹² are separately CO, SO₂, C═C(CN)₂, S, O or C(CH₃)₂;

each R¹³ is alkyl, branched alkyl or heterocyclic ring derivatized withcharged groups to enhance water solubility and enhance photostability;

R⁹ and R¹⁰ are chains carrying charged groups to enhance watersolubility (i.e. sulfonate, amide, ether) and/or chains bearing reactivegroups for conjugation to other molecules. The reactive group is afunctional group that is chemically reactive (or that can be madechemically reactive) with functional groups typically found inbiological materials, or functional groups that can be readily convertedto chemically reactive derivatives using methods well known in the art.In one embodiment of the invention, the charged and reactive groups areseparately haloacetamide (—NH—(C═O)—CH₂—X), where X is Cl, Br or I.Alternatively, the charged and reactive groups are separately amine,maleimide, isocyanato (—N═C═O), isothiocyanato(—N═C═S), acyl halide,succinimidyl ester, or sulfosuccinimidyl ester. In another embodiment,the charged and reactive groups are carboxylic acid (COOH), orderivatives of a carboxylic acid. An appropriate derivative of acarboxylic acid includes an alkali or alkaline earth metal salt ofcarboxylic acid. Alternatively, the charged and reactive groups arereactive derivatives of a carboxylic acid (—COORx), where the reactivegroup Rx is one that activates the carbonyl group of —COORx towardnucleophilic displacement. In particular, Rx is any group that activatesthe carbonyl towards nucleophilic displacement without beingincorporated into the final displacement product. Examples of COORx:ester of phenol or naphtol that is further substituted by at least onestrong electron withdrawing group, or carboxylic acid activated bycarbodimide, or acyl chloride, or succinimidyl or sulfosuccinimidylester. Additional charged and reactive groups include, among others,sulfonyl halides, sulfonyl azides, alcohols, thiols, semicarbazides,hydrazines or hydroxylamines.

For all dyes of the invention, any net positive or negative chargespossessed by the dye are balanced by a biologically compatiblecounterion or counterions. As used herein, a substance that isbiologically compatible is not toxic as used, and does not have asubstantially deleterious effect on biomolecules. Examples of usefulcounterions for dyes having a net negative charge include, but are notlimited to, alkali metal ions alkaline earth metal ions, transitionmetal ions, ammonium and substituted ammonium ions. Examples of usefulcounterions for dyes having a net positive charge include, but are notlimited to chloride, bromide, iodide, sulfate, phosphate, perchlorate,nitrate, tetrafluoroborate.

As used herein, R⁹ and R¹⁰ chains for conjugation are alkyl, ofunlimited length, preferably with 1-6 carbons, and can include othermoieties such as ether, amide, or sulfonate to improve water solubility.The groups can be substituted on the end or in the middle of the alkylchain.

In one embodiment, the fluorescent dye of the present invention has thefollowing structure (VI):

In another embodiment, the fluorescent dye of the present invention hasthe following structure (VII):

In another embodiment, the fluorescent dye of the present invention hasthe following structure (VIII):

Depending upon their environment, the flourescent dyes of the presentinvention can exist in somewhat different polarization states. Thisproperty can modulate the solubility and the emission wavelength of thedye. For example, the fluorescent dye depicted below can be charged ornon-charged.

The fluorescent dyes of the present invention can absorb and emit lightat a variety of wavelengths, depending on the arrangement and variety ofsubstituents employed. Thus, the choice of whether to use R⁸, R¹¹ andR¹² as —CO—, —SO₂—, —C═C(CN)₂—, —S—, —O— or —C(CH₃)₂— can influence theabsorption and emission wavelength. However, by varying the substituentsof the present invention, one of skill in the art can readily ascertainwhich combination of substituents will yield a fluorescent dye with adesired absorption and emission spectrum.

Moreover, according to the present invention, the degree of conjugationof the dye, and in particular, the length of the alkylene chainconnecting the two rings, can predictably influence the absorption andemission wavelength of the dye. Thus, addition of one —C═C— group canshift the fluorescence wavelength about +100 nm. Smaller incrementalchanges in the emission wavelength can be made by adding a conjugatedgroup to one of the rings in the dye. Thus, one of skill in the art canreadily modulate the emission wavelength of the dye as desired. In oneembodiment, the absorption and emission wavelengths can be altered torange from about 300 nm to about 800 nm. Preferably, the absorption andemission wavelengths of the present dyes range from about 450 nm tohigher wavelengths. Any variety of methods can be used to make thepresent dyes.

Preferred nucleic acid indicators include intercalating agents andoligonucleotide strands, such as, for example, YOYO-1, Propidium Iodide,Hoechst 33342, DAPI, Hoerchst 33258, SYTOX Blue, Chromomycin A3,Mithramycin, SYTOX Green, SYTX Orange, Ethidium Bromide, 7-AAD, AcridineOrange, TOTO-1, TO-PRO-1, Thiazole Orange, Propidium Iodide, TOTO-3,TO-PRO-3, LDS 751.

Synthon and Peptide Intermediates

The synthetic intermediates (i.e. synthons) of the invention thatinclude one or more aminooxy groups can be incorporated into peptidesusing a variety of techniques that are known in the art. For example, asdiscussed below, the synthons can be incorporated into a peptide usingsolid-phase peptide synthesis, solution-phase peptide synthesis, nativechemical ligation, intein-mediated protein ligation, and chemicalligation.

Peptides may be prepared using solid-phase peptide synthesis (SPPS). Forexample, according to the SPPS technique, protected amino acids inorganic solvents can be added one at a time to a resin-bound peptidechain, resulting in the assembly of a target peptide having a specificsequence in fully-protected, resin-bound form. The product peptide canthen be released by deprotection and cleavage from the resin support(Wade, L. G., JR., Organic Chemistry 4th Ed. (1999)). As illustrated inExample 2 below, amino acids containing an aminooxy functional group canbe incorporated into peptides using SPPS. Use of this methodology allowsan amino acid containing an aminooxy functional group to be positionedat a desired location within a synthesized peptide chain.

Amino acids containing an aminooxy group can also be incorporated into apeptide using solution-phase peptide synthesis (Wade, L. G., JR. OrganicChemistry 4th Ed. (1999)). Solution-phase peptide synthesis involvesprotecting the amino-terminus of a peptide chain followed by activationof the carboxyl-terminus allowing the addition of an amino acid or apeptide chain to the carboxy-terminus (Wade, L. G., JR. OrganicChemistry 4th Ed. (1999)).

Native chemical ligation is a procedure that can be used to join twopeptides or polypeptides together thereby producing a single peptide orpolypeptide having a native backbone structure. Native chemical ligationis typically carried out by mixing a first peptide with acarboxy-terminal α-thioester and a second polypeptide with anamino-terminal cysteine (Dawson, P. E., et al., (1994), Science266:776-779; Cotton, G. J., et al., (1999), J. Am. Chem. Soc.121:1100-1101). The thioester of the first peptide undergoesnucleophilic attack by the side chain of the cysteine residue at theamino terminus of the second peptide. The initial thioester ligationproduct then undergoes a rapid intramolecular reaction because of thefavorable geometric arrangement of the alpha-amino group of the secondpeptide. This yields a product with a native peptide bond at theligation site. A polypeptide beginning with cysteine can be chemicallysynthesized or generated by intein vectors, proteolysis, or cellularprocessing of the initiating methionine. This method allows mixing andmatching of chemically synthesized polypeptide segments.

The synthons of the invention are particularly useful in combinationwith native chemical ligation, because native chemical ligation allows asynthetic peptide having a specifically positioned amino acid (e.g. asynthon of the invention) to be selectively ligated to other peptides orinto a larger polypeptide, antibody or protein. The ability tospecifically incorporate aminooxy modified amino acids into a peptidechain allows useful moieties to be linked at any position within apeptide, polypeptide, antibody or protein. Examples of such moietiesthat can be incorporated into a peptide using this method include, butare not limited to, phosphorylated or glycosylated amino acids,unnatural amino acids, tags, labels, crosslinking reagents, biosensors,reactive groups, and fluorophores. Another advantage of native chemicalligation is that it allows incorporation of peptides into a polypeptidethat are unable to be added by ribosomal biosynthesis.

Intein-mediated protein ligation may also be used to selectively placeamino acids containing aminooxy functional groups into peptides. Inteinsare intervening sequences that are excised from precursor proteins by aself-catalytic mechanism and thereby expose reactive ends of a peptide.Intein vectors have been developed that not only allow single-steppurification of proteins, but also yield polypeptides with reactive endsnecessary for intein-mediated protein ligation (IPL) (also calledexpressed protein ligation)(EPL) (Perler, F. R. and Adam, E., (2000)Curr. Opin. Biotechnol. 11(4):377-83; and Evans, T. C., et al., (1998)Protein Sci 7:2256-2264). This method allows a peptide having aselectively placed amino acid containing an aminooxy functional group tobe readily ligated to any peptide with reactive ends generated by inteinexcision.

Two peptides or polypeptides may also be linked through use of chemicalligation. Chemical ligation occurs when two peptide segments are eachlinked to functional groups that react with each other to form acovalent bond producing a non-peptide bond at the ligation site (Wilken,J. and Kent, S. B. H., (1998) Curr. Opin. Biotechnol. 9:412-426). Thismethod can be used to ligate a peptide having a specifically positionedaminooxy functional group to another peptide or polypeptide to produce adesired polypeptide that may be later linked to a detectable group.

A functional molecule (“D”) can be attached to a peptide comprising anaminooxy group through a direct linkage (e.g. an amide bond—O—N—C(═O)-D) or through a linking group. The structure of the linkinggroup is not crucial, provided it does not interfere with the use of theresulting labeled peptide. Preferred linking groups include linkers thatseparate the aminooxy nitrogen and the detectable group by about 5angstroms to about 100 angstroms. Other preferred linking groupsseparate the aminooxy nitrogen and the detectable group by about 5angstroms to about 25 angstroms.

For example, the linking group can conveniently be linked to thedetectable group through an: 1) amide (—N(H)C(═O)—, —C(═O)N(H)—), 2)ester (—OC(═O)—, —C(═O)O—), 3) ether (—O—), 4) thioether (—S—), 5)sulfinyl (—S(O)—), or 6) sulfonyl (—S(O)₂) linkage. Such a linkage canbe formed from suitably functionalised starting materials usingsynthetic procedures that are known in the art.

The linking group can conveniently be linked to the nitrogen of theaminooxy group to form an amide (—O—N(H)C(═)— or a thiourea linkage(—O—N—C(═S)—N—) linkage, using reagents and conditions that are known inthe art.

The aminooxy group can be attached to a peptide through a direct bond(e.g. a carbon-oxygen bond) between the aminooxy oxygen and a side chainof the peptide, or the aminooxy group can be attached to the peptidethrough a linking group. The structure of the linking group is notcrucial, provided it does not interfere with the use of the resultinglabeled peptide. Preferred linking groups include linking groups thatseparate the aminooxy oxygen and the side chain of the peptide by about5 angstroms to about 100 angstroms. Other preferred linking groupsseparate the aminooxy oxygen and the side chain of the peptide by about5 angstroms to about 25 angstroms.

A specific linking group (e.g. X or Y) can be a divalent(C₁-C₆)alkylene, (C₂-C₆)alkenylene, or (C₂-C₆)alkynylene chain, or adivalent (C₃-C₈)cycloalkyl, or aryl ring.

Thus, a simple and efficient synthetic methodology for site-specificlabeling of peptides after synthesis has been developed that provideshigh yield, selectivity and compatibility with both solid-phase peptidesynthesis and C^(α)-thioester peptides. The approach and primaryadvantages can be summarized as follows:

(1) A protected aminooxy amino acid that can be incorporated intopeptides has been synthesized;

(2) Procedures have been optimized to yield highly efficient andspecific modification of the aminooxy nitrogen in the presence ofunprotected competing nucleophiles, including cysteine, lysine and aminogroups;

(3) One preferred electrophile that can be used for labeling, anactivated carboxylic ester, is readily available in the majority ofcommercially available fluorescent dyes and labels;

(4) Labeling of the aminooxy group occurs after synthesis andpurification, thus enabling the use of chemically sensitive fluorophoresand labels that would otherwise not survive earlier syntheticprocedures;

(5) The synthetic methodology is compatible with the steps required forthe synthesis of proteins by total chemical synthesis or expressedprotein ligation, namely synthesis of C^(α)-peptide thioesters andamide-forming ligations; and

(6) Combinatorial screening of both the functional molecule and itsplacement will enable the rapid synthesis of optimally labeledpolypeptide-based biosensors.

Biosensors

Using the methods of the present invention with the synthons andpeptides provided herein, antibodies, antigens and other polypeptidescan be labeled with or attached to functional molecules. Such labeled orconjugated antibodies and polypeptides have particular utility asbiosensors. As used herein, a “biosensor” is a peptide, antigen,polypeptide or antibody with an attached functional molecule. In oneembodiment, the functional molecule is a label or dye, however, as usedherein, a biosensor can have any functional molecule attached thereto.For example, the functional molecule can be a label, dye, biophysicalprobe, peptide, polynucleotide, therapeutic agent, pharmaceutical,toxin, cross-linking group (chemical or photochemical), a compound thatmodifies the biological activity of the peptide, or a caged molecule(e.g. a reporting molecule or a biologically active agent that is maskedand that can be unmasked by photoactivation or chemical means).

In one embodiment the functional molecule is dye or label. Such a dye orlabel can be an environmentally-sensitive fluorescent dye such thatfluorescence is emitted by the dye can change when the protein biosensorbecomes exposed to a different environment. Such a change in environmentcan occur, for example, when the biosensor binds to, or associates with,a target protein or cellular structure. The change in fluorescence canbe used to quantify or otherwise monitor the amount of binding orinteraction between the biosensor and the target site. The Examplesprovided herein further illustrate how such a biosensor can be used.

The present invention provides methods for identifying an optimalposition for the functional molecule on the peptide, polypeptide orantibody which involve generating a series of peptides, each peptidehave the same amino acid sequence and functional molecule. However, thefunctional molecule is positioned at a different location along thebackbone of each peptide in the series. To determine which location isbest, the strength of the signal from the various peptides is observedunder different conditions and the optimal location provides optimalfunctioning by the functional molecule and the peptide, polypeptide orantibody. For example, when the functional molecule provides a signal, astronger signal is preferred so long as the function and the chemicaland physical properties of the peptide, protein or antibody are notimpaired. When an environmentally sensitive functional molecule ischosen, a maximal change in signal is preferred as the environment ischanged. For example, when the environmentally sensitive functionalmolecule is attached to detect an interaction of the peptide with atarget, a maximal change in signal is preferred when the peptide bindsor interacts with its target, unless of the location of the functionalmolecule affects the binding affinity, binding selectivity or anotherdesirable attribute of the peptide.

Thus to determine an optimal location for a functional molecule in anantibody or polypeptide which can bind to a target, a series of peptidesare first synthesized, each with the functional molecule at a differentposition. The peptides can be incorporated into the polypeptide orantibody using the methods described herein to generate a series ofbiosensors, each with a functional molecule at a somewhat differentposition. The interaction of the different polypeptide or antibodybiosensors with target is observed. In general, an optimal position forthe functional molecule on such a biosensor is that position whichpermits stable and selective binding to target with a maximal signalchange upon binding.

However, some variability in binding and signal strength can betolerated so long as an observable, localized signal is detected uponinteraction of the biosensor and the target. Thus, the strength ofbinding between the biosensor and target should be sufficient to permitobservation of bound biosensor. If the biosensor is only transientlybound, little or no localized signal from the probe may be observed;instead, only diffuse signal from biosensor in solution may be observed.Similarly, if the biosensor is bound non-selectively, non-localizedsignal may be detected from many sites. Obviously, a strongly localizedsignal that clearly correlates with biosensor binding to target ispreferred. A readily detectable change in signal strength or quality asthe biosensor and target interact is also preferred.

Procedures known to one of skill in the art can be used to detect asignal provided by the functional molecule and to correlate a change insignal by an environmentally sensitive functional molecule. Signalscontemplated by the present invention include fluorescent emissions,radioactive emissions, enzymatic production of a colored product, andthe like. One of skill in the art can readily detect these signals usinga fluorescence microscope, a scintillation counter, a light orradioactively sensitive photoemulsion, a light microscope, aspectrophotometer or other means. When an environmentally-sensitivefunctional molecule is used, the signal can change in lifetime,strength, color or other quality to signal interaction of the functionalmolecule with the environment. For example, when theenvironmentally-sensitive functional molecule is a fluorescent dye, thesignal change can be a color, wavelength, intensity or lifetime offluorescence emitted by the dye. The change need only be detectable, forexample, a change in wavelength of fluorescence of about 50 nm to about300 nm can be readily detected. However, a change in wavelength ofgreater than about 10 nm is preferred. More preferably the change inwavelength is greater than 20 nm. In one embodiment the change inwavelength can vary from about 30 nm to about 200 nm.

Where convenient, a peptide, as opposed to a polypeptide or antibody,with an attached functional molecule may be a biosensor, for example,when the binding affinity and selectivity of the peptide isrepresentative of the larger protein of which it is normally a part.Alternatively, the labeled peptide can be ligated into a polypeptide toform the protein or antibody of which it is normally a part using themethods described herein. In one embodiment, the peptide is incorporatedonto one of the termini of a protein, for example, through proceduresdescribed in Curr. Op. Biotechnology 9:412 (1998) or Ann. Rev. Biochem.57:957-89 (1988). In another embodiment, the peptide can be incorporatedinto the middle of a protein, for example, by using the proceduresdescribed in PNAS 95:6705 (1998).

Targets

As used herein, a “target” is any molecule, structure or complex that apeptide, polypeptide or antibody linked to a functional molecule by thepresent methods can interact with. Targets contemplated by the presentinvention include antigens, antibodies, proteins, enzymes, membraneproteins, endoplasmic reticulum proteins, structural proteins, majorhistocompatibility proteins, DNA binding proteins, receptors, ligands,cofactors, nucleic acids, kinases, GTPases, ATPases, proteins involvedin motility and the like. Targets may undergo structural changes andother types of changes, for example, phosphorylation,de-phosphorylation, conformational changes, ligand binding, co-factorbinding, activation, post-translational modification, carbohydrate orsugar attachment, membrane interactions and the like. Specific targetsinclude calmodulin, Rho GTPases, rac, cdc42, mitogen-activated proteinkinase (MAP kinase), Erk1, Erk2, Erk3, Erk4, IgE receptor (F_(c)□RI)actin, α-actinin, myosin, and major histocompatibility proteins.

Targets can have “tag” sequences that permit binding of a biosensor orlabeled polypeptide to the target. As used herein, a “tag” is anybinding site for a biosensor. In general, a tag is a peptide orpolypeptide segment that is recognized by the biosensor and to which thebiosensor will bind with sufficient binding affinity to permit detectionand/or visualization of the target. Any protein binding domain or ligandbinding site of a receptor can be used. Thus, a tag can be, for example,an antigenic epitope to which an antibody can bind, a part of aleucine-zipper to which another part of a leucine zipper can bind, adomain of a homeotic protein that mediates binding to a DNA site,receptor-ligand binding site.

The dimerizing domain from yeast GCN4 protein can also be used to form atag-biosensor dimer. The GCN4 dimerizing domain is a single α-helixwhich pairs with its partner to form a parallel coiled-coil (O'Shea,Rutkowski, Stafford and Kim, Science, 245, 646, (1989)). The X-raycrystal structure of the GCN4 coiled-coil has been determined (O'Shea,Klemm, Kim and Alber, Science, 254, 539, (1991)), and factors affectingstability and oligomerization state of native and mutant GCN4coiled-coil peptides have been determined (Harbury, Zhang, Kim andAlber, Science, 262, 1401, (1993); O'Neil, Hoess and DeGrado, Science,249, 774, (1990)). In addition, several model coiled-coil peptides havebeen extensively studied and rules governing stability of these designedpeptides have been reported (Hodges, Zhou, Kay and Semchuk, PeptideRes., 3, 123, (1990); Zhu et al., Int. J. Peptide Protein Res., 40, 171,(1992); Lumb and Kim, Biochemistry, 34, 8642, (1995)).

Another useful tag-biosensor dimer can be formed from the binding domainfrom the Arc repressor of bacteriophage P22 (Schildbach, J F, Milla M E,Jeffrey P D, Raumann B E, Sauer R T (1995). Biochemistry 34: 13914-19).Arc repressor is a dimeric polypepfide of 53 amino acids. Atag-biosensor dimmer can also be formed from the C-terminaltetramerizing domain from the tumor suppressor p53. The structure ofthis domain has been determined from the crystal Jeffrey, Gorina andPavletich, Science, 267, 1498, (1995)) and in solution (Clore et al.,Nature Struct. Biol., 2, 321, (1995)). The Mnt repressor ofbacteriophage P22 (Waldburger, C. D. and R. T. Sauer (1995).Biochemistry 34(40): 13109-13116), the Mnt repressor polypeptide andstreptavidin-avidin can be used to form tag-biosensor dimers.Streptavidin (Argarana, C., Kuntz, I. D., Birken, S, Axel, R. andCantor, C. R. (1986) Nucleic Acids Res. 14:1871) and avidin (Green, N.M. (1975) Adv. Protein Chem. 29:85) are homologous tetramericpolypeptides of approximately 125-127 and 128 amino acids, respectively.Biotin ligase (BLS) can also be used to make a tag-biosensor dimmer. BLSpolypeptide sequences have been described for various proteins: forexample, the C-terminal 87 residues of the biotin carboxy carrierprotein of Escherichia coli acetyl-CoA carboxylase (Chapman-Smith, A.,D. L. Turner, et al. (1994). Biochem J. 302: 881-7). The C-terminal 67residues of carboxyl-terminal fragments of human propionyl-CoAcarboxylase alpha subunit may be used (Leon-Del-Rio, A. and R. A. Gravel(1994) J. Biol. Chem. 269(37): 22964-8); and residues 18-123 ofPropionibacterium freudenreichii transcarboxylase 1.3S biotin subunit(Yamano, N., Y. Kawata, et al. (1992) Biosci. Biotechnol. Biochem.56(7): 1017-1026).

Such tag sequences can be present naturally or can be added to thetarget by methods available to one of skill in the art. Differenttargets within a single cell can have different tags to which differentbiosensors can bind. This permits visualization of two or more targetswithin the same cell so that the interaction and dynamic interplaybetween targets can be observed in relation to other cellularstructures. As described herein, biosensors can also be designed toundergo fluorescence resonance energy transfer (FRET) when they becomejuxtaposed at an appropriate distance. Similarly, a target can beengineered to have two different tags so that two different biosensorscan bind to the same target. Tag sequences can be added to a targetprotein, for example, by protein ligation procedures described herein orby standard molecular biology techniques where a peptide or polypeptideis fused to another peptide or polypeptide. See, e.g., Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A LaboratoryManual, 2nd ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

Antigen and Antibody Targets

When antibodies are linked to functional molecules using methods of thepresent invention the target can be an antigen. Alternatively, anantigen or a peptide epitope can serve as a biosensor for an antibodytarget. The present invention contemplates use or detection of anyantigen or antibody known to one of skill in the art as a target.

In general, a molecule must have sufficient complexity and sufficientmolecular weight in order to act as an antigen. In order to havesufficient complexity, the antigen must have at least one epitope. Asused in this invention, the term “epitope” is meant to include anydeterminant capable of specific interaction with the monoclonalantibodies of the invention. Epitopic determinants usually consist ofchemically active surface groupings of molecules such as amino acids orsugar side chains and usually have specific three dimensional structuralcharacteristics, as well as specific charge characteristics.

In order to have a sufficient molecular weight, the antigen generallymust have a molecular weight that is greater than 2,000 daltons.Formerly, it was thought that the lower molecular weight limit to conferantigenicity was about 5,000 daltons. However, antigenicity has recentlybeen demonstrated with molecules having molecular weights as low as2,000 daltons. Molecular weights of 3,000 daltons and more appear to bemore realistic as a lower limit for immunogenicity, and approximately6,000 daltons or more is preferred.

In preparing antigens to produce antibodies for attachment to functionalmolecule, it is desirable to use antigens with a high degree of purity.Accordingly, it is desirable to use a purification process permittingisolation of the antigen from antigenically distinct materials.Antigenically distinct materials are undesired large molecules that maycompete with the target antigen for antibody production therebyminimizing production of the desired antibodies or inducingcross-reactive antibodies of low specificity or affinity. The practiceof the invention can accordingly include a number of purification stepsusing available techniques. Purification can, for example, be effectedby size exclusion chromatography, ion exchange chromatography, dialysis,cold organic solvent extraction, gel electrophoresis and/or fractionalcrystallization means which are available to one of skill in the art.Preferably an antigen used for antibody preparation is at least about90% pure and more preferably at least about 99% pure.

Removal of small molecule reactants and reaction products from thesynthesized antigen is generally desirable. However, some smallmolecular substances may be useful, for example, for control of pH andsalinity. Thus, a convenient end-product form in which to recover theantigen is, in a buffered aqueous solution that is suitable for directadministration to animals.

Antibodies

The present invention contemplates linkage of any available antibody tofunctional molecules. Such antibodies can be polyclonal or monoclonalantibodies. The term “antibody” as used in this invention is meant toinclude intact antibody molecules as well as fragments thereof, such as,for example, Fab and F(ab′)₂, which are capable of binding to theantigen or its epitopic determinant.

Polyclonal antibodies can be raised by administration of an antigen ofthe invention to vertebrate animals, especially mammals such as goats,rabbits, rats or mice using known immunization procedures. Usually abuffered solution of the antigen accompanied by Freund's adjuvant isinjected subcutaneously at multiple sites. A number of suchadministrations at intervals of days or weeks is usually necessary. Anumber of animals, for example from 3 to 20, are injected with theexpectation that only a small proportion will produce desirableantibodies. However, one animal can provide antibodies sufficient forthousands of assays. Antibodies are recovered from animals after someweeks or months.

Exemplary immunogenic carrier materials can be used with the antigen toenhance the immune response. The carrier material can be a natural orsynthetic substance, provided that it is an antigen or a partialantigen. For example, the carrier material can be a protein, aglycoprotein, a nucleoprotein, a polypeptide, a polysaccharide, alipopolysaccharide, or a polyamino acid. See also, the carrier moleculesand antibody production methods set forth in Cremer et al., “Methods inImmunology” (1963), W. A. Benjamin Inc., New York, pp. 65-113 and Harlowand Lane, “Antibodies: A Laboratory Manual” (1988), Cold Spring HarborLaboratory, New York, p. 5. These disclosures are herein incorporated byreference.

A preferred class of natural carrier materials is the proteins. Proteinscan be expected to have a molecular weight in excess of 5,000 daltons,commonly in the range of from 34,000 to 5,000,000 daltons. Specificexamples of such natural proteins are hen ovalbumin (OA), bovine serumalbumin (BSA), keyhole limpet hemocyanin (KLH), horse gammaglobulin(HGG), and thyroglobin.

Exemplary of synthetic carrier is the polyamino acid, polylysine. Wherethe synthetic antigen comprises a partially antigenic carrier conjugatedwith a hapten, it will generally be desirable for the conjugate to havea molecular weight in excess of 6,000 daltons, although somewhat lowermolecular weights may be useful. Preferably, the natural carrier hassome solubility in water or aqueous alcohol. Desirably, the carriers arenontoxic to the animals to be used for generating antibodies.

The carrier can be coupled to antigens by any available means, includedthe procedures provided herein. Preferably, a carrier moiety has aplurality of hapten or antigen moieties coupled to it, for example,about 15 to 30 for a protein of 100,000 daltons. While steric hindranceand reduced structural complexity may reduce the number of haptens orantigens attached to the carrier, the maximum number is preferred. Forexample, up to about 25 to about 50 hapten moieties can be coupled toBSA carriers.

The use of monoclonal antibodies as biosensors of this invention ispreferred because monoclonal antibodies are homogeneous and can becontinuously produced in large quantities. Monoclonal antibodies areprepared by recovering lymph node or spleen cells from immunized animalsand immortalizing the cells in conventional fashion, e.g., by fusionwith myeloma cells or by Epstein-Barr virus transformation. Clonesexpressing the desired antibody are identified by screening cell linemedia for reactivity with the antigen used to immunize the animals. Oneof skill in the art can use readily available methods to make monoclonalantibodies, for example, by using the hybridoma technique describedoriginally by Koehler and Milstein, Eur. J. Immunol., 6:511 (1976), byHammerling et al., in “Monoclonal Antibodies and T-Cell Hybridomas”,Elsevier, N.Y., pp. 563-681 (1981), and by Zola, in “MonoclonalAntibodies: A Manual of Techniques”, CRC Press, Boca Raton, Fla. (1987).The hybrid cell lines can be maintained in vitro in cell culture media.The cell lines producing the antibodies by these procedures can beselected and/or maintained in a medium containinghypoxanthine-aminopterin thymidine (HAT). However, once the hybridomacell line is established, it can be maintained on a variety ofnutritionally adequate media. Moreover, the hybrid cells lines can bestored and preserved in any number of conventional ways, includingfreezing and storage under liquid nitrogen. Frozen cell lines can berevived and cultured indefinitely with resumed synthesis and secretionof monoclonal antibody.

The secreted antibody is recovered from tissue culture supernatant orascites fluid by conventional methods such as immune precipitation, ionexchange chromatography, affinity chromatography such as proteinA/protein G column chromatography, or the like. The antibodies describedherein are also recovered from hybridoma cell cultures by conventionalmethods such as precipitation with 50% ammonium sulfate. If desired, thepurified antibodies can then be sterile filtered before use.

The term “monoclonal antibody” as used herein refers to any antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations, which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Inaddition to their specificity, the monoclonal antibodies areadvantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins.

The monoclonal antibodies herein also include hybrid and recombinantantibodies produced by splicing a variable (including hypervariable)domain of an anti-adduct antibody with a constant domain (e.g.“humanized” antibodies), or a light chain with a heavy chain, or a chainfrom one species with a chain from another species, or fusions withheterologous proteins, regardless of species of origin or immunoglobulinclass or subclass designation, as well as antibody fragments (e.g., Fab,F(ab′)₂ and Fv), so long as they exhibit the desired biologicalactivity. See e.g. Cabilly et al. U.S. Pat. No. 4,816,567; Mage andLamoyi, “Monoclonal Antibody Production Technique and Applications”, pp.79-97 (Marcel Dekker, Inc., New York, 1987).

Thus, the modifier “monoclonal” indicates the character of the antibodyas being obtained from a substantially homogenous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method described by Koehler and Milstein, supra,or may be made by recombinant DNA methods (Cabilly, et al. supra).

In one embodiment, the biosensor antibodies or the present invention areused for diagnostic or imaging purposes in vivo, within a mammaliansubject. While the in vivo use of a monoclonal antibody from a foreigndonor species in a different host recipient species is usuallyuncomplicated, an adverse immunological response by the host toantigenic determinants present on the donor antibody can sometimesarise. In some instances, this adverse response can be so severe as tocurtail the in vivo use of the donor antibody in the host. Further, theadverse host response may serve to hinder the intercellularadhesion-suppressing efficacy of the donor antibody. Methods to avoidsuch adverse reactions are available. For example, humanized antibodiesor chimeric antibodies (Sun, et al., Hybridoma, 5 (Supplement 1):S17,1986; Oi, et al., Bio Techniques, 4(3):214, 1986) can be used. Chimericantibodies are antibodies in which the various domains of theantibodies' heavy and light chains are coded for by DNA from more thanone species. Typically, a chimeric antibody will comprise the variabledomains of the heavy (V_(H)) and light (V_(L)) chains derived from thedonor species producing the antibody of desired antigenic specificity,and the constant domains of the heavy (C_(H)) and light (C_(L)) chainsderived from the host recipient species. It is believed that by reducingthe exposure of the host immune system to the antigenic determinants ofthe donor antibody domains, especially those in the C_(H) region, thepossibility of an adverse immunological response occurring in therecipient species will be reduced. Thus, for example, it is possible toproduce a chimeric antibody for in vivo clinical use in humans whichcomprises mouse V_(H) and light V_(L) domains coded for by DNA isolatedfrom ATCC HB X, and C_(H) and C_(L) domains coded for with DNA isolatedfrom a human leukocyte.

The present invention further provides a composition comprising anantibody with a functional molecule attached by the methods of thepresent invention and a suitable carrier. Further, the present inventionalso provides a therapeutic composition comprising an effective amountof the antibody-functional molecule conjugate produced by the presentmethods and a pharmaceutically acceptable carrier.

As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutically accepted carriers, suchas phosphate buffered saline solution, water, emulsions such as anoil/water emulsion or a triglyceride emulsion, various types of wettingagents, tablets, coated tablets and capsules. An example of anacceptable triglyceride emulsion useful in intravenous andintraperitoneal administration of the compounds is the triglycerideemulsion commercially known as Intralipid R™. Typically such carrierscontain excipients such as starch, milk, sugar, certain types of clay,gelatin, stearic acid, talc, vegetable fats or oils, gums, glycols, orother known excipients. Such carriers may also include flavor and coloradditives or other ingredients.

Methods of Using Biosensors

Biosensors of the invention can be used in vitro or vivo. Biosensors canbe used with any type of test sample, such as cultured cells, tissuesamples, cell suspensions, cell lysates, partially purified isolates ofa potential target and purified isolates of a potential target. A samplethat includes cells can be in any form convenient for observation of thetarget, for example, cells plated on a culture dish, cells on amicroscope slide, suspensions of cells, tissues in physiological orculture media or tissues on a microscope slide

In general, the biosensor is contacted with a sample that may contain atarget of interest under conditions and for a time sufficient to permitinteraction or binding of the biosensor to the target of interest. Thebiosensor can be contacted with sample that is in solution by simplymixing the biosensor into the solution. When the target is in a cell ortissue, the biosensor can be injected into the cell or tissue. In someexperiments placing the needle into the region just adjacent to thenucleus produced a good combination of efficient injection and cellhealth. Alternatively, the cell or tissue can be transfected ortransformed with a nucleic acid capable of expressing the biosensor. Oneof skill in the art can use readily available procedures for injectingand transfecting cells with such biosensors.

Conditions sufficient to permit interaction of the biosensor with targetare generally conditions that permit protein-protein interactions. Atemperature sufficient to permit protein-protein interactions is atemperature that maintains and encourages secondary and tertiarypolypeptide structure formation. For example, the temperature can beabout 4° C. to about 40° C., and preferably a temperature of about 4° C.to about 37° C. When the sample includes cells, the temperature ispreferably a temperature which maintains cellular function, for example,a temperature of about 20° C. to about 38° C. A time sufficient forinteraction of the biosensor with a target can be determined by one ofskill in the art. Such a time can be, for example, about 1 minute toabout 2 hours, preferably about 3 minutes to about one hour. In oneexperiment, visualization of biosensor-target interaction wassuccessfully performed after cells were given about 5-10 minutes torecover after injection.

The invention will now be illustrated by the following non-limitingExamples.

EXAMPLE 1 Site-Specific Labeling of a Secondary Aminooxy Group

Under controlled pH conditions, the low pKa and enhanced nucleophilicityof an aminooxy group relative to other nucleophilic side chains found inpeptides suggested the possibility of site-specific reaction withstandard electrophiles such as succimidyl esters (FIG. 1). Whileselective labeling of a primary aminooxy group in the context of anunprotected peptide was achieved, extensive attempts to utilize theprimary aminooxy group during synthesis failed. Even when protected asthe 2-chlorobenzyloxycarbonyl carbamate, deprotonation of the primaryaminooxy group allowed rapid acylation, so it could not be readilyincorporated during peptide synthesis. Thus, under certain conditions,the use of a secondary aminooxy group may be preferred.

A test peptide containing both a secondary aminooxy group andnucleophilic amino acids that were most likely to interfere withselective labeling at the aminooxy nitrogen (lysine, cysteine, and theamino-terminus) was prepared. As illustrated in FIG. 2,NH₂-AKAARAAAAK*AARACA-CO₂H (SEQ ID NO: 2), here designated SA-testpeptide, was synthesized by incorporation and deprotection ofN-(2-Cl-benzyloxycarbonyl)-N-methylaminooxy acetic acid (FIG. 4, 3)during solid phase peptide synthesis. The reactivity of the protectedsecondary aminooxy group was sufficiently attenuated to remainunreactive during Boc solid-phase peptide synthesis on thioester-linkerresins. The 2-Cl-Z protection for the N-methylaminooxy amino acid wasefficiently removed by standard HF cleavage procedures.

Conditions for selectively labeling the secondary aminooxy group weredetermined by varying the reaction pH and dye stoichiometry. Labelingwith the succinimide ester of tetramethylrhodamine (TMR—OSu) wasdetermined to be optimal in a solvent system consisting of 50% DMSO/50%aqueous acetate buffer at pH of 4.7 with 2 equivalents of dye per moleof peptide (Table 1). The crude reaction products were separated fromunreacted dye, and characterized by RP—HPLC and ESI-MS. Under theseconditions, a single molecule of dye was incorporated on the aminooxygroup with a 78% yield, based on HPLC quantification. However, sidereaction products were also isolated and determined to be either SA-testpeptide labeled with two dye molecules (˜13%) or acetylated peptideproducts (˜5%). The selectivity, as defined by the ratio of the peakareas of desired single-labeled product over double-labeled products,was 6/1 (Table 1). TABLE 1 Labeling of SA-Test Peptide Buffers Gel DyePercentage Reaction Filtration TCEP pH* Equivalents SM 1 Dye 2 DyeSelectivity Acetate (NH₄)HCO₃ No 4.7 1.0 32 56 7 8:1 Acetate (NH₄)HCO₃No 4.7 1.5 14 63 13 4.5:1   Acetate (NH₄)HCO₃ No 4.7 2.0 0 78 13 6:1Acetate (NH₄)HCO₃ No 4.7 2.4 0 76 17 4.5:1   Acetate (NH₄)HCO₃ No 4.73.0 0 71 23 3:1 Citrate 0.1% TFA Yes 4.7 4.3 4.3 89.6 4.1 22:1 Carbonate 0.1% TGA Yes 9.0 0.99 13 85 0  51:1**

It was determined that many of the side reactions occurred during sizeexclusion chromatography in the ammonium bicarbonate solvent used toseparate reaction products. Using an acidic solvent system, 0.1% TFA,virtually eliminated multiply labeled side products leading toconsiderable improvements in both selectivity and yield. Including amild reducing agent, tris(2-carboxyethyl)phosphine (TCEP), in thereaction buffer also significantly curtailed several minor sidereactions revealed by HPLC, especially disulfide formation. Labeling andgel filtration under these optimized conditions produced a 70% recoveredyield of labeled SA-test peptide (90% yield based on HPLC quantitation),90% of which was labeled with only a single dye at the aminooxy amine.The labeling selectivity was increased to 22/1 (Table 1).

The purity of the product was confirmed using RP—HPLC, massspectroscopy, further chemical reaction, and isolation of purposefullyoverlabeled products. The labeled peptide eluted as a single peak underall HPLC conditions tested with a mass consistent with that predictedfor singly-labeled SA-test peptide (Mass=1970). To determine that thelabeling site was indeed at the N-methylaminooxy group, a selectivezinc/acetic acid reduction procedure was used to cleave the N—O bond(FIG. 3). HPLC of the reduction reaction showed >98% conversion of thestarting material and a new earlier eluting peak. The mass of this peak(1530 amu) corresponded to the predicted mass of the unlabeled SA-testpeptide cleaved at the aminooxy N—O bond. The residual zinc was washedseveral times with a saturated solution of EDTA in water, whichdemonstrated that the reduction reaction was complete.

To eliminate the unlikely possibility that the HPLC peak containingisolated single-labeled SA-test peptide product was a mixture of twolabeled species, SA-test peptide was reacted under higher pH conditions(pH 9.0) to label all reactive sites. SA-test peptide contained 3nucleophilic labeling sites which would be irreversibly labeled:aminooxy, lysine, and N-terminal amine. Dye labeling at high pHgenerated a mixture of peptides labeled at all possible combinations ofsites with 1, 2 or 3 dyes. HPLC analysis of this reaction mixture showed8 peaks, indicated by ESI-MS to correspond to unreacted SA-test peptide,SA-test peptide single-labeled at the aminooxy nitrogen, and sixadditional peaks corresponding to two single-labeled peptides, threepeptides bearing two dyes and a single triply-labeled peptide species.This experiment revealed the HPLC retention times of all these products,none of which co-eluted with the peak identified as SA-test peptidelabeled with a single dye on the secondary aminooxy nitrogen.

Site-specific labeling of the aminooxy group could even be achieved atbasic pH (Table 1). Using pH 9.0 carbonate buffer in our solvent system,addition of 0.5 equivalents of dye produced, after 3 hours, ˜50%conversion of the starting SA-test peptide to a single peak with theelution time of the desired singly-labeled product. After addition ofanother 0.5 equivalents of TMR—OSu and an additional 3 hours of reactiontime, HPLC showed ˜85% conversion to a peak with the retention time ofthe desired product. Two minor peaks (˜2-3% of total peak area), werealso apparent and corresponded to the two other single-labeled SA-testpeptide species identified in the multiple labeling experiment above.The N-hydroxysuccinimide ester of rhodamine clearly showed selectivereactivity with the aminooxy group.

The selectivity observed at higher pH cannot be explained by thenucleophilicity of the aminooxy group alone. In fact, others have shownthat at, in an uncatalyzed reaction with phenyl acetate at high pH,amines are more reactive than O-alkyl aminooxy groups. Therefore wesuggest that kinetic factors are contributing to the selectivereactivity of the N-methylaminooxy group, even when competing groups arenot protonated. Possible reasons for this include: (1) the aminooxyoxygen localizes the nitrogen near the activated ester via formation ofa hydrogen bonded “bridged” intermediate (2) a base catalyzed reactionpathway under the conditions of our reaction. This exceptionalreactivity has important practical implications, as it can allow theselective labeling of acid-labile polypeptides and synthetic proteinsunder physiological or basic conditions.

EXAMPLE 2 The Secondary Aminooxy Group is Compatible withC^(α)-Thioesters and Amide-Forming Ligation

Preparation of proteins by total chemical synthesis often requires theligation of large polypeptides prepared by solid-phase peptide synthesison thioester-linker resins. The most generally applicable methodsavailable for ligations are native chemical ligation and expressedprotein ligation. These processes utilize the same basic chemistry tojoin two peptides, one with an N-terminal cysteine and the other with aC-terminal thioester, through a regiospecific and site-specific reactionto generate a larger polypeptide. The application of aminooxy-labelingchemistry to the synthesis of large polypeptides and proteins requirescompatibility with these solid-phase peptide synthesis and ligationchemistries.

The optimal approach for utilizing aminooxy-labeling chemistry in thechemical synthesis of proteins is direct incorporation of the aminooxygroup as part of an amino acid used during standard solid-phase peptidesynthesis. For this purpose, we generated a suitably protectedN-methylaminooxy amino acid,α-Boc-β-[N-(2-Chlorobenzyloxycarbonyl)-N-MethylaminooxyAcetyl]-α,β-Diaminopropionic Acid [Boc-2-Cl-Z-(SA)Dapa-OH] (4), as shownin FIG. 4. This amino acid, referred to as SAOD, was incorporated intothe peptide sequence LY—(SAOD)-AG-MPAL thioester by synthesis on TAMPALthioester-linker resin, as described below in the Methods. (MPAL is theC-terminal mercaptopropionyl-leucine group generated by cleavage of apeptide from TAMPAL resin, see Hojo, H., et al., Bull. Chem. Soc. Jpn.1993, 66:2700-2706; and Hackeng, T. M. et al., Proc. Natl. Acad. Sci.USA. In press)

Ligation of the LY—(SAOD)-AG-MPAL (SEQ ID NO: 3) thioester peptide withthe peptide CRANK—NH₂ (SEQ ID NO: 4), was tested using standardprocedures employing phosphate buffer with 6M guanidine hydrochloride atneutral pH in the presence of 2-3% thiophenol by volume. The ligationproceeded over 24 hours and generated the desired ligation product,LY—(SAOD)-AGCRANK—NH₂ (SEQ ID NO: 5), at ˜85% yield. The major sideproduct was attributable to modification of unligated CRANK—NH₂ peptideunder the ligation conditions (mass=714.5, data not shown), and was notrelated to the presence of the aminooxy group. There was also a singletime-dependent side reaction, which generated a product of 14 mass unitslower than the desired. Using high concentrations of reacting peptidesand isolating the ligation product after 24 hours reduced this sidereaction to acceptable levels (<5%).

The ligation of a peptide containing multiple potentially reactivefunctional groups, including a hexahistidine tag useful for affinitychromatography was also tested. CouplingCEYRIDRVRLFVDKLDNIAQVPRVGAA-HHHHHH (SEQ ID NO: 6) to LY—(SAOD)-AG-MPALthioester proceeded to completion in 5 hours with minimal sidereactions. In both ligation reactions, there was less than 1%LY—(SAOD)-AG-MPAL self condensation product, indicating that theaminooxy group and thioester do not appreciably react with one anotherunder the ligation conditions. These results demonstrate that theinclusion of an unprotected aminooxy group in the peptide chain iscompatible with native chemical ligation.

Labeling of the two ligation products using tetramethylrhodaminesuccinimide ester proceeded with selectivity similar to that for theSA-test peptide. HPLC integration indicated that the product of theLY—(SAOD)-AG-MPAL ligation with CRANK—NH₂ was labeled with greater that95% efficiency and with a selectivity of 34:1. Mass spectral analysisand zinc reduction demonstrated labeling at only the aminooxy group. Forthe longer hexahistidine-containing polypeptide ligation product,selectivity for the aminooxy group was greater than 10:1, but it wasdifficult to achieve high yields. The histidines could potentially havebeen affecting yield and selectivity by catalyzing nucleophilic attackon the succinimide ester of the reactive dye. Inclusion of guanidinehydrochloride in the reaction solvent increased the yield toapproximately 50%, indicating that folding or poor solubility of thepeptide was a factor in preventing access of the reactive dye to theaminooxy group. Selectivity was also improved, presumably because of theavailability of the reactive secondary aminooxy group. Single-sitelabeling at the aminooxy group was proven by mass spectral analysis oftrypsin and α-chymotrypsin digests of the labeled polypeptide product.

EXAMPLE 3 Specificity of Labeling in Protein Domains Containing AminooxyAmino Acids

As a control to establish the selectivity of labeling for aminooxy aminoacid, the labeling of native β-Lactoglobulin with tetramethylrhodamineN-hydroxysuccinimide ester was attempted. It was found that non-specificlabeling of this 162 aa protein containing 15 lysines and 4 cysteineswas minimal (<1%), even after 6 hours.

Finally, the GTPase binding domain of p21 activated kinase (45 aa, 4lys, 1 cys) with a secondary aminooxy amino acid incorporated at theamino-terminus (SAOD-PBD) was prepared. Previous experiments havedemonstrated that PBD domains labeled with fluorescent reporter dyes atthis terminus could be used as biosensors of GTPase activation. Labelingof PBD using the new methodology would enable the production ofsufficient quantities to apply the biosensors in vivo and inpharmaceutical screening applications, and would allow incorporation ofsensitive detectable groups enabling applications within living cells.

SAOD-PBD was readily labeled with Alexa-532 N-hydroxy-succinimide esterby titration addition of dye at pH 4.7 over 72 hours. The labelingefficiency was commensurate with that reported for the longest modelpeptide (50% yield by HPLC quantitation) and there was no indication ofmultiple labeling. In this case, isolation of labeled SAOD-PBD byRP—HPLC proved difficult. Separation to baseline resolution was notachieved, but small quantities of unlabelled PBD in the labeled productdo not preclude the use of the labeled material in biosensorapplications. Previous reports indicate that separation of labeledproduct from starting polypeptide is highly dependent on the specificpeptide and the attached dye.

These results demonstrate that the optimized site-specific labelingchemistry reported here is compatible with the steps required for thepreparation of proteins by total chemical synthesis.

EXAMPLE 4 Materials and Methods

-   General: For column chromatography, silica gel (230-400 mesh) was    used in standard glass columns with gravity or air pressure.    Reversed-phase high performance liquid chromatography (RP—HPLC) was    performed on a Waters HPLC system with UV detection at 214 nm using    either a Vydac C-18 analytical column (5 μm, 0.46×25cm), a Waters    RCM 8×10 module equipped with a semi-preparative Delta Pack C-18    Radial Pack cartridge column (15 μm, 8×100 mm) from Millipore, or a    Vydac C-18 preparative scale column (15 μm, 1.0×25 cm). Linear    gradients of solvent B (0.09% TFA in 90% acetonitrile/10% water) in    solvent A (0.1% TFA in water) were used for all HPLC chromatographic    separations.

Mass spectra of peptides were obtained either with a Sciex API-IIIelectrospray ionization (ESI) triple-quadrupole mass spectrometer (PEBiosystems, Foster City), or matrix assisted laser desorption ionizationtime-of-flight (MALDI-TOF) instruments from Thermo Bioanalysis (ThermoBioanalysis, LTD., UK) or Kratos Analytical (Chestnut Ridge, NY.). ForESI—MS, the observed masses reported were derived from the experimentalm/z states for all observed charge states of a molecular species usingthe program MacSpec (Sciex, version 2.4.1) for electrospray massspectrometry. MALDI—MS observed masses were relative to internalcalibration using α-cyano-hydroxycinnammic acid or sinipinic acidmatrices. Calculated masses reported were derived from either MacProMass(Terry Lee and Sunil Vemuri, Beckman Research Institute, Duarte, Calif.)or PAWS (Version 8.1.1, ProteoMetrics) and reflect the average isotopecomposition of the singly-charged molecular ion. Proton nuclear magneticresonance spectrometry was recorded on a Bruker AC-250 mass spectrometerand data was analyzed using WinNMR (Bruker Instruments).Ultraviolet-Visible spectroscopy was performed on a Hewlett-Packardphotodiode-array spectrophotometer.

Boc-L-amino acids were purchased from Novabiochem (La Jolla, Calif.) orBachem Bioscience, Inc. (King of Prussia, Pa.).[[4-(Hydroxymethyl)phenyl]-acetamido]methyl (—OCH₂-Pam) Resin waspurchased from PE Biosystems (Foster City, Calif.) andmethylbenzhydrylamine (MBHA) resin was purchased from PeninsulaLaboratories, Inc. (San Carlos, Calif.). Solvents were Synthesis gradeor better and were purchased from Fisher Scientific (Tustin, Calif.).Trifluoroacetic acid (TFA) and anhydrous hydrogen fluoride werepurchased from Halocarbon (New Jersey) and Matheson Gas (RanchoCucamonga, Calif.). Dyes were obtained from Molecular Probes (Eugene,Oreg.). All other reagents were analytical grade or better and werepurchased from Aldrich (Milwaukee, Wis.), Lancaster (Windham, N.H.),Peptides International (Louisville, Ky.) or Richelieu Biotechnologies(Montreal, Canada).

Peptide Segment Synthesis. Synthesis of peptides was carried outmanually using optimized stepwise solid-phase synthesis methods with insitu neutralization and HBTU activation procedures for Boc chemistry oneither —OCH₂-Pam, MBHA, or Trt-protected mercaptopropionyl-Leu (TAMPAL)resin (Hojo, H., et al., Bull. Chem. Soc. Jpn. 1993, 66:2700-2706;Hackeng, T. M. et al., Proc. Natl. Acad. Sci. USA. In press; andSchnolzer, M., et al., Int. J. Peptide Protein Res. 1992, 40:180-193).Standard Boc protecting group strategies were employed. Coupling wasmonitored by quantitative ninhydrin assay after 15 minute couplingcycles. After chain assembly, standard deprotection and cleavage fromthe resin support was carried out by treatment at 0° C. for 1 hour withanhydrous HF containing either 10% p-cresol or anisole as scavenger.Purification was performed using RP—HPLC.

Synthesis of TAMPAL Resin (Hojo, H., et al., Bull. Chem. Soc. Jpn. 1993,66:2700-2706). 2.5 grams of MBHA resin (0.865 mmol/g, 2.16 mmol ofamine) was swelled in DMF. Boc-Leu-OH (1.1 grams, 4.4 mmol) wasactivated with HBTU (8 ml, 0.5M solution) and DIEA (2 ml ), then coupledto the MBHA resin until complete reaction by ninhydrin assay. TheN^(α)-Boc group of the linked leucine was removed with neat TFA, thenS-Trt-β-mercaptopropionic acid (1.5 grams, 4.3 mmol), activated in thesame manner as Boc-Leu-OH, was added to the deprotected Leu-MBHA resinand allowed to couple until complete reaction. TheS-Trt-β-mercaptopropionyl-Leu-MBHA resin was washed extensively withDMF, then DCM/MeOH (1/1), and finally dried in vacuo to yield 3.39 gramsof thioester resin. Substitution calculated by weight gain yielded 0.549mmol/gram.

Deprotection of TAMPAL Resin: S-trityl protection was removed by two 5minute treatments with 95% TFA/5% triisopropylsilane. The deprotectedresin was extensively with DMF before coupling the first amino acid,activated using optimized in situ neutralization protocols.

Synthesis of N-(2-Cl-benzyloxycarbonyl)-N-Methylhydroxylamine (1)(Jencks, W. P., Carriuolo, J. J. Am. Chem. Soc. 1960, 82:675; Jencks, W.P. J. Am. Chem. Soc. 1958, 80:4581, 4585). N-methylhydroxylaminehydrochloride (0.95 g, 11.37 mmol) was dissolved in 3 ml of water withrapid stirring. The pH of this solution was adjusted to 6-7 by dropwiseaddition of a saturated solution of sodium bicarbonate.2-Chlorobenzyloxycarbonyl-N-hydroxysuccinimidyl carbonate (1.2 g, 4.23mmol) was dissolved in 4 ml of THF and added slowly to the rapidlystirring solution of neutralized N-methylhydroxylamine. After stirringat room temperature for 14 hours, the reaction was quenched with 20 mlof saturated sodium bicarbonate and extracted three times with 20 mlethyl acetate. The combined ethyl acetate layers were washed once withsaturated sodium bicarbonate, dried over anhydrous sodium sulfate andthe solvent was removed in vacuo to yield 0.77 g (3.77 mmol, 84%) of anoff-white solid. TLC Rf=0.2 (Hex/EtOAc/AcOH 80/20/1). ¹H NMR: 3.23(s,3H), 5.25 (s, 2H), 7.24 (m, 2H), 7.37 (m, 2H). HRMS: Expected=216.0427,Observed=216.0425.

Synthesis of N-(2-Cl-benzyloxycarbonyl)-N-Methylaminooxy AceticAcid-Tert-butyl ester (2) (Jerry March in Advanced Organic Chemistry,Third Edition. John Wiley & Sons, New York. 1989, pp 381; and Nyberg, D.D., Christensen, B. E. J. Am. Chem. Soc. 1957, 79:1222; Motorina, I. A.,et al., Synlett 1996, 389). Compound 1 (0.96 g, 4.71 mmol) was dissolvedat room temperature in 10 ml of THF with rapid stirring. Bromoacetatetert-butyl ester (1.05 g, 5.38 mmol) was added, then sodium iodide (1.5g, 10.01 mmol) followed by DIEA (2.5 ml, 15.92 mmol). The reactionchanged to an orange-yellow color after addition of sodium iodide. Thereaction was quenched with 30 ml water after complete reaction (˜3hours) and extracted 3 times with ethyl acetate. The combined ethylacetate layers were dried over sodium sulfate and the ethyl acetate wasremoved in vacuo. The resultant oily solid was purified by silicachromatography on 230-400 mesh silica gel using hexanes/ethylacetate/acetic acid (80/20/1) to yield 1.40 g (4.29 mmol, 90%) of a pureyellow oil. TLC Rf=0.5 (Hex/EtOAc/AcOH 80/20/1). ¹H NMR: 1.46 (s, 9H),3.29 (s, 3H), 4.36 (s, 2H), 5.26 (s, 2H), 7.24 (m, 2H), 7.40 (m, 2H).HRMS: Expected Mass=330.1108, Observed Mass=330.1104.

Synthesis of N-(2-Cl-benzyloxycarbonyl)-N-Methylaminooxy Acetic Acid (3)(Bryan, D. B., et al., J. Am. Chem. Soc. 1977, 99:2353). Compound 2 (1.1g, 3.30 mmol) was dissolved into 4 ml of DCM and, with rapid stirring,neat TFA (5 ml) was added dropwise over 2 minutes at room temperature.After 1 hour, the reaction was quenched with 20 ml water, extracted 3times with DCM, and the combined DCM layers were dried over sodiumsulfate. The DCM was removed in vacuo to yield 0.9 g (3.28 mmol, 99%) ofan off-white solid. TLC Rf=0.2 (Hex/EtOAc/AcOH 80/20/1). ¹H NMR: 3.23(s, 3H), 4.50 (s, 2H), 5.32 (s, 2H), 7.28 (m, 2H), 7.40 (m, 2H). HRMS:Expected Mass=274.0482, Observed Mass=274.0479.

Synthesis ofN-(2-Cl-benzyloxycarbonyl)-N-Methylaminooxyacetyl-α-Boc-α,β-DiaminopropionicAcid [(SA)Dapa-OH] (4) (Wahl, F., Mutter, M. Tett. Lett. 1996,37:6861-6864; and Anderson, G. W., et al., J. Am. Chem. Soc. 1964,86:1839). N-(2-Chlorobenzyloxycarbonyl)-N-Methylaminooxy Acetic Acid (3)(2.5 g, 9.2 mmol) was activated with N-hydroxysuccinimide (2.11 g, 2equiv.) and DIC (1.440 ml, 1.0 equiv.) in 20 ml DCM. This reaction wasrapidly stirred at room temperature for 2 hours prior to the addition ofN^(α)-Boc-α,β-diaminopropionic acid (2.3 g, 1.2 equiv.) and DIEA (3.20ml, 2 equiv.). After 4 hours, the DCM solvent was removed in vacuo, and50 ml ethyl acetate was added. The ethyl acetate layer was washed twicewith 0.5M acetate buffer, pH=4.0, then twice with 0.1N sulfuric acid.The combined acid washes were then washed with 50 ml ethyl acetate. Thecombined ethyl acetate layers were dried over sodium sulfate, thenconcentrated in vacuo to yield a viscous yellow oily solid. This solidwas subjected to 3 hexane precipitations from diethyl ether to yield2.16 g (51% yield) of an off-white solid. TLC Rf=0.2-0.4 (Hex/EtOAc/AcOH30/70/0.5). 1H NMR: 1.45 (s, 9H), 3.16 (s, 3H), 3.54 (d-of-t, 1H,J=14.3, 4.6Hz), 3.93 (m, 1H, J=14.3, 7.5, 4.6 Hz), 4.31 (s, 0.5H), 4.38(s, 1H), 4.45 (s, 1H), 4.51 (s, 0.5H), 5.34 (s, 2H), 5.97 (broad-d, 1H,J=7.3 Hz), 7.30 (m, 2H), 7.43 (m, 2H), 8.50 (broad-s, 1H). HRMS:Expected Mass=460.1487, Observed Mass=460.1480.

Synthesis of Secondary Aminooxy Test Peptide (SA-test peptide). TheSA-test peptide, NH₂-AKAARAAAAK*AARACA-CO₂H, was synthesized with Lys 10side chain Fmoc protection as described previously (Canne, L. E., etal., J. Am. Chem. Soc. 1995, 117:2998-3007). Incorporation of thesecondary aminooxy group was accomplished by coupling 2-Cl-Z protectedN-methylaminooxyacetic (300 mgs, 1.09 mmol) activated withDiisopropylcarbodiimide (157 ul, 1.00 mmol) and N-hydroxysuccinimide(140 mgs, 1.22 mmol) in 2 ml DCM for 1-2 hours, hen diluted with 2 mlDMF just prior to coupling to the ε-amino group of Lys 10. Optimizedcoupling, cleavage and purification protocols were utilized. Amino acidanalysis was consistent with the desired peptide. Expected Mass=1560,Observed Mass=1559.

Synthesis of LY—(SAOD)-AG-MPAL-Thioester. LY—(SAOD)-AG-MPAL-Thioesterwas synthesized using optimized in situ neutralization protocols for Bocchemistry on TAMPAL resin. Coupling of the N^(α)-Boc-(SA)Dapa-OH aminoacid was accomplished by reacting the in situ activatedN-hydroxysuccinimide ester to the deprotected amino-terminal nitrogen ofalanine (Canne, L. E., et al., J. Am. Chem. Soc. 1995, 117:2998-3007).(SA)Dapa-OH (4) (230 mgs, 0.5 mmol) was dissolved in 1 ml DCM andN-hydroxysuccinimide (115.1 mgs, 1.0 mmol) and DIC (74.4 μl, 0.47 mmol)were added. The reaction was mixed briefly and allowed to activate for1-2 hours at room temperature prior to coupling to the deprotectedN-terminus of the peptide chain. After this coupling, no furthermodifications of the synthetic protocols were required. Expectedmass=797, Observed mass=797.

Ligation of LY—(SAOD)-AG-MPAL-Thioester with CRANK—NH₂ Peptide.LY—(SAOD)-AG-MPAL-Thioester (3 mg, 3.8 μmol) was dissolved into 100 ulof 50 mM phosphate buffer containing 6M guanidine hydrochloride, pH=7.2.To this solution was added CRANK—NH₂ peptide dissolved into 100 μl ofthe same phosphate buffer and 3 ul of thiophenol. The reaction wasmonitored by analytical reversed-phase HPLC. After 24 hours, the ligatedproduct, LY—(SAOD)-AGCRANK—NH₂ (SEQ ID NO: 10), was isolated bysemi-preparative reversed-phase HPLC (gradient=10-50% B over 60 minutes)and lyophilized to yield a fluffy white solid. Amino acid analysis wasconsistent with the desired product peptide. Expected Mass=1168,Observed Mass=1168.

Ligation of LY—(SAOD)-AGMPAL-Thioester withCEYRIDRVRLFVDKLDNIAQ-VPRVGAA-HHHHHH (SEQ ID NO: 7). LY—(SAOD)-AG-MPAL(0.3 mgs, 0.37 mmol) and CEYRIDR-VRLPFVDKLDNIAQ-VPRVGAA-HHHHHH (1.5 mgs,3.8 mmol) were subjected to the same ligation and purificationconditions as described above to yield 1.0 mgs (58% yield) of a whitefluffy solid. Expected Mass=4518, Observed Mass=4517.

Synthesis of Amino-Terminal P21 Binding Domain (PBD) Peptide Fragment,(SAOD)-KKKEKERPEISLPSDFEHTIHVGFDA-MPAL Thioester (SEQ ID NO: 8):Secondary aminooxy containing amino-terminal PBD thioester weresynthesized as described above using TAMPAL resin. HF cleavage utilizingp-Cresol scavenger followed by HPLC purification yielded(SAOD)-KKKEKERPEISLPSDFEHTIHVGFDA-MPAL containing two DNP groupsprotecting the histidines. Mass Expected=3745, Mass Observed=3745.

Synthesis of Carboxy-Terminal of P21 Binding Domain (PBD) PeptideFragment, CTGEFTGMPEQWARLLQT (SEQ ID NO: 9): The native carboxy-terminalhalf of PBD was synthesized using standard FMOC synthesis protocols bythe Scripps Peptide and Protein Core Facility. Mass Expected=2068, MassObserved=2068.

Synthesis of SAOD-Modified PBD,SAOD-KKKEKERPEISLPSDFEHTIH-VGFDACTGEFTGMPEQWARLLQT (SEQ ID NO: 11): 1.5mg of SAOD-KKKEKERPEISLPSDFEHTIHVGFDAMPAL (0.4 mmol) was ligated to 1 mg(4.8 mmol) of carboxy-terminal fragment, CTGEFTGMPEQWARLLQT, asdescribed above. After 48 hours, the ligated PBD proteins were isolatedby RP—HPLC and lyophilized, 1.5 mg (70% yield), Mass Expected=5262, MassObserved=5262.

Selective Labeling of SA-Test Peptide with TetramethylrhodamineN-hydroxysuccinimidyl ester. A solution of SA-test peptide (3.396 μg/μl,2.18 mM) in 5% acetate buffer, pH=4.7 incorporating 5 mM TCEP wasutilized for labeling. A stock solution of dye (5 μg/μl, 9.5 mM) wasmade by dissolving TMR—OSu in neat DMSO. For each reaction, the dyestock was diluted so that the desired number of dye equivalents could beadded in 20 μl of DMSO. The following equivalents of dye were tested:1.2, 1.5, 1.8, 2.0, 2.4, 3.0, and 4.3. With constant stirring, 20 μl ofdye solution was added in two 10 μl aliquots to 20 μl of peptidesolution at room temperature. The second aliquot of dye in DMSO wasadded 10 minutes after the initial dye addition. After complete additionof dye, the reaction was briefly vortexed, then incubated at roomtemperature. After 3 hours, the labeled reaction product(s) wereseparated from unreacted dye by gel filtration on Sephadex G-10 or G-15columns using either 100 μM ammonium bicarbonate or, after optimization,0.1% TFA in water. The individual peptide product(s) were then separatedby RP—HPLC and analyzed by ESI-MS. Mass Expected=1971, Mass Observed1970.

Non-Selective Labeling of SA-Test Peptide with TetramethylrhodamineN-hydroxysuccinimide ester. SA-test peptide (3.396 μg/μl, 2.18 mM) wasdissolved 100 mM sodium carbonate, pH=9.01 containing 5 mM TCEP. 18 μlof neat DMSO was added to 20 μl of peptide solution. With rapid mixing,1 μl stock dye solution in DMSO was added to this mixture. Uponcompletion of addition, the reaction was vortexed briefly, thenincubated at room temperature. After 3 hours, a 1 μl aliquot was removedand evaluated by RP—HPLC and ESI-MS. This process was repeated untilsignificant levels of reaction were detected by formation ofsingle-labeled SA-lysine test peptide products.

Selective Labeling of LY—(SAOD)-AGCRANK—NH₂ andLY—(SAOD)-AGCEYRIDRVR-LFVDKLDNIAQVPRVGAA-HHHHHH withTetramethylrhodamine N-hydroxy-succinimidyl Ester. A sample of 20 μL ofLY—(SAOD)-AGCRANK—NH₂ (2.5 μg/μl, 2.18 mM) in 200 mM citrate buffer,pH=4.7, 5mM TCEP was labeled using 4.3 equivalents of dye in DMSO,purified and analyzed. Expected Mass=1580, Observed Mass=1580. A 20 μLsample of LY—(SAOD)-AGCEYRIDRVRLFVDKLDNIAQVPRVGAA-HHHHHH (SEQ ID NO: 12)(9.7 μg/μl, 2.12 mM) in 200 mM citrate buffer, pH=4.7, containing 5 mMTCEP and 3M guanidine hydrochloride was labeled using a modifiedprocedure. 18 μl of a solution of tetramethylrhodamineN-hydroxysuccinimide (10 μg/μl) in DMSO was added in 6 μl aliquots over15 minutes with rapid mixing. The reaction was incubated at roomtemperature for 5 hours prior to gel filtration/RP—HPLC purification andmass spectral analysis. Expected Mass=4930, Observed Mass=4929.

Labeling of P-Lactoglobulin with TetramethylrhodamineN-hydroxysuccinimide ester: 10 μL of a 10 mg/ml solution oftetramethylrhodamine N-hydroxysuccinimide ester in DMSO (9.5 equivalentscompared to protein), was added to a solution containing 10 μL of DMSOand 20 μL of a solution of β-Lactoglobulin (20.3 mg/ml or 1.1 mM) in 2.8M guanidine hydrochloride with 5 mM TCEP (pH 4.7). After 3 hours,protein was separated from unreacted dye by gel filtration, and labelingwas determined by analysis of the dye-to-protein ratio (proteinconcentration was determined by the method of Waddel and ε fortetramethylrhodamine in phosphate buffer, pH=8.0 of 81,000).

Labeling of PBD Proteins with Alexa-532 N-Hydroxysuccinimide Ester: 150μg of the SAOD-modified PBD protein was dissolved in 105 μL of 200 mMsodium citrate buffer, pH=4.8, containing 5 mM TCEP (proteinconcentration ˜0.28 mM). A solution of Alexa-532-OSu in DMSO (dyeconcentration ˜10 mg/ml in DMSO) was titrated into the protein solutionin 5 μL aliquots over 72 hours. Four hours after each addition, theextent of labeling was determined by RP—HPLC and MS. Labeling wascontinued until quantities of double-labeled PBD was obtained(SAOD-modified PBD). Alexa-532 labeled SAOD-modified PBD, MassExpected=5871, Mass Observed=5870.

Zinc/Acetic Acid Reduction of the N-methylaminooxy N—O Bond in Peptides.Reductive cleavage of the N—O bond was performed using zinc and aqueousacetic acid. Effervescence in the reaction was evident after a fewseconds and subsided after 60-120 minutes. After 14 hours, the reactionsupernatant was analyzed by RP—HPLC and ESI-MS. Reduction of labeledSA-Test peptide, Expected Mass=1530, Observed Mass=1530: Reduction oflabeled (SAOD)-AGCRANK—NH₂: Expected Mass=1139, Observed Mass=1139.

Trypsin/Chymotrypsin Cleavage of LabeledLY—(SAOD)-AGCEYRIDRVRLFVDKLDNIAQVPRVGAAHHHHHH Peptide. 10 μl of a 0.05mg/ml solution of either trypsin or α-chymotrypsin in 25 mM ammoniumcarbonate (without pH adjustment) was added to 5 μl of a 10-20 μg/μlsolution of pure tetramethylrhodamine-labeled peptide in water (finalconcentration of protease is 0.033 mg/ml). The reaction was incubated atroom temperature for 24 hours prior to analysis of peptide fragments byMALDI-MS.

EXAMPLE 5 Synthesis and Characterization of Fluorescent Dyes

Materials and Methods. Commercially available solvents and reagents werepurchased from major suppliers. ¹H NMR spectra of dye solutions inDMSO-d₆ were recorded at 500.11 Hz on Bruker Avance DRX-500 MHz. Thepentet corresponding to the residual protons of DMSO-d₆ (2.49 ppm) wasused as internal reference.

The analytical sample of the dyes was purified by HPLC on a Vydac C-18column (no. 218TP152022, 22*250 mm, 15-20 μm, 3 mL/min) usingacetonitrile-water gradient elution.

Absorbance spectra were recorded on Hewlett-Packard HP8452 diode arrayspectrophotometer and fluorescence spectra were recorded on SPEXFluorolog 2 spectrometer.

All reactions were run in a oven-dried round bottom flask, under argonatmosphere, and capped with a rubber septum. 1-Benzothiophen-3(2H)-one1,1-dioxide was synthesized according the procedure of M. Regitz (Chem.Ber., 1965, 98: 36). 3-(dicyanomethylene)indan-1-one was synthesizedfrom 1,3-indandione by the method of K. A. Bello, L. Cheng and J.Griffiths (Chem. Soc. Perkin Trans. II, 1987, 815-818).

Preparation of2Z)-2-[(2E)-3-METHOXYPROP-2-ENYLIDENE]-1-BENZOTHIPHEN-3(2H)-ONE1,1-DIOXIDE (Compound 1-SO),(2Z)-2-[(2E)-3-METHOXYPROP-2-ENYLIDENE]-3-(DICYANOMETHYLENE)INDAN-1-ONE(Compound 1-CN)

To a solution of corresponding ketone (2.5 mmol) in 20-50 ml of methanolwas added trifluoroacetic acid in one aliquot at 80° C. followedimmediately by the addition of 2 ml of 1,3,3-trimethoxy propene. Thereaction mixture turned into a clear dark brown solution and after aninterval of 30 sec a yellow solid precipitated out. The precipitate wasfiltered and dried.

For compound 1-SO, the yield was 60%. 1H NMR (CDCl₃): δ 4.04 (s, 3H,CH₃O), 6.53 (t, ³J_(H—H)=12.1 Hz, 1H), 7.53 (d, ³J_(H—H)=11.7 Hz, 1H),7.74 (d, ³J_(H—H)=12.8 Hz, 1H), 8.2-8.4 (m, 4 H, Ph).

For compound 1-CN, the yield was 60%. ¹H NMR(CDCl₃): δ 8.67 (dd, 6.2 Hz,2.9 Hz, 1H), 8.37 (d, 11.7 Hz, 1H), 7.87 (m, 1H), 7.73 (m, 3H), 7.51 (d,12.05 Hz, 1H), 3.98 (s, 3H).

PREPARATION OF(2Z)-2-[(2E,4E)-4-(3-(3-SULFONATOPROPYL)-1,3-BENZOTHIAZOL-2(3H)-YLIDENE)BUT-2-ENYLIDENE]-1-BENZOTHIOPHEN-3(2H)-ONE1,1-DIOXIDE (DYE 2)

1.16 g (2.00 mmol) of3-(2-methyl-1,3-benzothiazol-3-ium-3-yl)propane-1-sulfonate and 625 mg(2.50 mmol) of compound 1-SO were dissolved in 65 ml of mixtureCH₃OH—CHCl₃ (1:1) and heated at reflux. 328 mg (4.00 mmol) of AcONa in10 ml was added at once and the reaction mixture was refluxed foradditional 30 min. After cooling, the dye was separated by filtration,recrystallized from methanol and dried. The yield was 65%.

¹H NMR: (DMSO-d₆) δ 1.85 (p, ³J_(H—H)=6.2 Hz, 2H, CH₂—CH₂—CH₂), 2.60 (t,³J_(H—H)=6.2 Hz, 2H, CH₂—CH₂—SO₃), 4.45(t, ³J_(H—H)=6.2 Hz, 1H, CH₂—N),6.87 (d, ³J_(H—H)=13.2 Hz, 1H), 7.3-8.1 (m, 11H). TABLE 2 SelectedMerocyanine Dyes. Com- pound Dye Structure 1

2

3

4

TABLE 3 Spectral Data for Selected Merocyanine Dyes.¹ ExtinctionAbsorption Emission Quantum Compound coefficient (*10⁻³) Maximum MaximumYield 1 100 618 636 0.07 2 100 603 623 0.10 3 120 586 618 0.17 4 80 667693 0.10¹All spectral data are recorded in n-butanol.

EXAMPLE 6 Imaging the Spatio-Temporal Dynamics of Rac Activation in vivowith FLAIR

FLAIR (Fluorescent Activation Indicator for Rho GTPases) is a biosensorsystem that maps the spatial and temporal dynamics of Rac activation inliving cells. The approach is based on microinjection of a fluorescentlylabeled domain from p21-activated kinase into cells expressing GFP-Rac.The injected domain (called PBD, for p21-binding domain) binds only toRac-GTP, and not to Rac-GDP (Thompson et al., 37 Biochemistry 7885(1998); Benard et al., 274 J. Biol. Chem. 13198 (1999)). Within livingcells, PBD binds to the GTP-Rac wherever it has bound GTP, bringing theAlexa546 dye on the PBD near the GFP on the Rac to produce fluorescenceresonance energy transfer (FRET). Thus, the FRET signal markssubcellular locations where Rac is activated. This can be quantified tofollow the changing levels and locations of Rac activation or to tracethe kinetics of total Rac activation on an individual cell basis.

The labeling of PBD with Alexa, and mammalian expression vectors forexpression of Rac-GFP is by any procedure, for example, as described inthis application. This example provides protocols for production of purePBD, protocols for generating cell images suitable for quantitativeanalysis of rac activation, and procedures and caveats for generatingtwo types of data: images showing the spatial distribution of Racactivation within cells, and curves showing the kinetics of Racactivation in single cells.

PBD Expression and Purification. PBD was expressed in the form ofC-terminal 6His fusion from the prokaryotic expression vector pET23(Novagen). It was determined experimentally that the highest levels ofexpression are observed when a vector containing plain T7 promoter (notT7lac) is used in combination with a BL21(DE3) strain (not the morestringent BL21(DE3)pLysS) of E. coli. This system allows for “leaky”protein expression (Novagen). While the 6His tag can be cleaved from thepurified protein with thrombin, it is not necessary, as the tag does nothave any significant effect on probe functionality.

Competent BL21 (DE3) cells (Stratagene) are transformed with pET23-PBDusing standard procedures. See, e.g., Sambrook et al., MolecularCloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press 1989)After transformation, cells were plated on an LB plate containingcarbenicillin. Cells do not degrade carbenicillin as quickly asampicillin, so a higher percentage of cells retain the vector at theculture density appropriate for induction (Novagen). Five ml of LB mediawith 100 μg/ml carbenicillin were inoculated with a single colony ofcells, and grown in the shaker at 37° C. for 6-8 hours (until dense).Two ml of this are then used to inoculate 50 ml of LBcarb. The rest ofthe culture is diluted 1:1 with glycerol and frozen for long termstorage at −80° C. The 50 ml culture is incubated in the shakerovernight at 37° C. Next morning 1-2 L of LBcarb are inoculated with theovernight culture (15-20 mL culture/500 mL media), and grown in theshaker (37° C.) to OD₆₀₀=0.8-0.9 (about 2-3 hours). After that thecultures are briefly chilled on ice to 30-32° C., then put back in theshaking incubator turned down to 30-32° C. The protein is expressed at alowered temperature to increase the portion of the correctly folded,soluble PBD. IPTG is added to a final concentration of 0.4-0.5 mM, andthe cultures are allowed to grow for another 4-5 hours at 30-32° C.(shaker). The cells are collected by centrifugation (8,000 G, 4 min),and stored as a pellet at −20° C. until use. Approximately 2.5-3 g ofcells is usually obtained from each liter of culture.

Purification of PBD-6His is performed essentially as described in theTalon affinity resin manual (Clontech). The cells (3-5 g) are thawed in20-30 ml of the Lysis buffer (30 mM Tris HCI, pH 7.8, 250 mM NaCl, 10%glycerol, 5 mM MgCl₂, 2 mM β-ME, 1 mM PMSF), homogenized with a spatulaand sonicated (4 pulses, 10-15 sec each). T4 lysozyme and DNAse areadded in catalytic amounts (approximately 100 micrograms/ml lysozyme and500 U DNAse) to help the lysis, and the suspension is incubated on icewith periodic mixing for 30 min. The cells are then centrifuged at12,500 rpm for 30 min, and the supernatant containing PBD is carefullytransferred into a 50 mL Falcon tube.

Talon resin (1.5-2 ml) (Clontech) is washed twice with 10 volumes of thelysis buffer in a 15 ml Falcon tube, centrifuging in the swinging bucketcentrifuge at low speed in between to separate the resin. The celllysate is added to the 1.5 ml of washed Talon resin in a 50 mL falcontube, and inverted or agitated gently (i.e. with an orbit shaker) for20-30 mm at r.t. The resin is then separated by centrifugation in aswinging bucket centrifuge. The supernatant containing the unboundfraction is removed and saved for SDS-PAGE analysis. The resin is thentransferred into a new 15 mL Falcon tube and washed twice (10-15 mineach, r.t., orbit shaker) with 12 mL of the lysis buffer, without PMSFand βME. The third wash is performed with lysis buffer+10 mM imidazole(add 1 M stock in water, kept at −20° C.). After the final separation,the resin is resuspended in 2-3 mL of lysis buffer with 10 mM imidazole,and pipetted into a column (0.5 cm in diameter). The resin is allowed tosediment by gravity flow until the fluid above the resin bed is almostgone, and then another 3-5 mL of Lysis buffer with 10 mM imidazole isadded to wash the column. The elution is performed using Lysis bufferwith 60 mM imidazole, and ca. 500 μL fractions are collected. PBDusually elutes in fractions 5-13 (total volume about 3-4 mL). An aliquotof each fraction is run on a 12% SDS-PAGE and the fractions containingthe pure PBD are combined and dialyzed against 1 L of 25 mM NaP buffer(pH 7.3). A dialysis bag (SpectraPor 7), or dialysis cassette (Pierce)with a molecular weight cutoff value of 3,500 kDa can be used.

After 2-3 hours of dialysis, the bag is wiped with a KimWipe and buriedin Aquacide powder (Caibiochem) for 15-45 min at 4° C., depending on thevolume of the sample in the bag. This concentration process should bemonitored carefully as complete drying may occur if the bag is left inthe Aquacide for too long. The powder is scraped gently from the bagevery 10-15 min to facilitate water absorption. When the sample reaches0.5-1.5 mL in volume (3-10-fold concentration), the Aquacide is cleanedfrom the bag, and the sample is carefully removed. The sample is brieflycentrifuged (14,000 rpm, 2 min) to separate it from the precipitatedmaterial, and transferred into a new dialysis bag or cassette. After thesecond dialysis step, the concentration of PBD is measured by taking asmall aliquot (5-10 μL) and diluting into 50 mM TrisHCl (pH 7.5-8.0) orother appropriate buffer. The extinction coefficient of PBD at 280 nm is8,250 (estimated from the primary sequence). On average, 1.5-2 mg of PBDis obtained per liter of cell culture.

Other methods of concentrating PBD were found to be less effective. Forinstance, centrifugal concentrators require prolonged centrifigations,and result in nonspecific adsorption of the small PBD protein to themembrane. It is essential to perform dialysis after concentration withAquacide. This prevents the ionic strength of the resultant protein prepfrom becoming too high before labeling. Low ionic strength conditionsare preferable to avoid excessive precipitation of the protein duringattachment of the hydrophobic dye.

Loading GFP-Rac and Alexa-PBD in cells. Cells were first transfectedwith GFP-Rac through nuclear microinjection. The EGFP variant (Clontech)was used that produced significantly brighter cells than wild type GFP(Heim et al., 6 Current Biology 178 (1996)). For microinjection of DNAand of PBD-Alexa, glass pipettes with 1.0 mm outer diameter and 0.50 mminner diameter (Sutter) were pulled using a micropipette puller (SutterModel P-87) to make microinjection needles with tips of approximately0.5 μm diameter. Rac-GEP c-DNA is injected into Swiss 3T3 fibroblasts at200 ng/μL, using a constant needle pressure of approximately 100 hPa.DNA can be centrifuged prior to injection (20,000 G for 15 minutes) toprevent clogging the needle.

Cells expressing GFP-Rac were microinjected with Alexa-PBD using amicroscope with optics and illumination capable of revealing the GFPfluorescence (detection sensitivity is typically improved by usinghigher NA objectives and brighter light sources, such as a 100 W Hg arclamp). Thus, only GFP-expressing cells need to be injected. To reducebackground fluorescence during injection and the following experiment,cells are placed in 1 mL of pre-warmed Dulbecco's Phosphate BufferSolution (DPBS) containing 1000 mg/L D-glucose, 36 mg/L sodium pyruvateand supplemented with 0.2% BSA, 1% L-glutamine and 1%Penicillin-Streptomycin. During injection and the following experiment,cells are mounted in a Dvorak live cell chamber (Nicholson) preheated to37° C., and maintained at 37° C. by a heated stage (20/20 Technology).The microscope can be equipped with a motorized stage and shuttercontrols (Ludi) to monitor multiple stage positions in one experiment.

Cells that are barely expressing or expressing too much GFP-Rac wereignored. The former produce FRET too weak for recording, and in thelatter overexpressed Rac affects the biology of the cell. In general, weobserved that cells expressing less than 300 intensity units (IU) do notdisplay Rac-induced ruffling and altered morphology. The precise valueof this cutoff will depend on the sensitivity of the imaging system, andshould be determined by each lab for a relevant biological behavior.

A 100 μM solution of Alexa-PBD was centrifuged at 20,000 G for 1 hourprior to injection, and then injected into the cytoplasm of cellsexpressing the GEP-Rac. Lowering the needle into the region justadjacent to the nucleus seems to produce the best combination ofefficient injection and cell health. After the injection, cells areplaced back into the 37° C. incubator for 5-10 minutes to recover.Alexa-PBD could potentially act as an inhibitor of Rac activity, socontrols were carried out showing that, for our imaging system, up to1000 IU of Alexa-PBD do not inhibit induction of ruffling.

Imaging Rac activation. Imaging experiments were performed using aPhotometrics KAF 1400 cooled CCD camera, and Inovision ISEE software forimage processing and microscope automation. Although filters areundergoing further optimization, the best success to date has beenachieved with the following filters, designed with sharp cutoffsspecifically for this purpose by the Chroma corporation: GFP: HQ480/40,HQ 535/50, Q505LP FRET: D480/30, HQ610/75, 505DCLP Alexa: HQ545/30,HQ610/75, Q565LP.

The exact camera settings depend on the type of experiment beingperformed. When the total Rac activity within the cell is beingdetermined, images are not generated, so spatial resolution can besacrificed for increased sensitivity. Images are taken using 3×3 binningwith exposure times of 0.1 sec, 0.1 sec, and 0.5 sec for GFP, Alexa andFRET respectively. When images are required, i.e. to examine thechanging spatial distribution of Rac activation, 1×1 binning is usedwith exposure times of 1 sec, 1 sec and up to 5 sec for GFP, Alexa andFRET respectively. These settings depend on the sensitivity of theimaging system used, and the desired trade off between sensitivity andspatial or temporal resolution. Settings should always be chosen notexceed the dynamic range of the camera (Berland et al., in Sluder et al.(eds.), Video Microscopy at 33 (Academic Press 1998). Motion artifactsshould also be considered during imaging experiments. For fast movingphenomena, features of the cell may move appreciably during the timebetween acquisition of the FRET and GFP images. This results inartifacts when the image is corrected for bleedthrough, as described inmore detail below. Such artifacts can be prevented by reducing the timebetween exposures, or by using two cameras simultaneously.

When total Rac activation is being determined, a picture of GFP-Rac andFRET is taken at each time point. Only one image of Alexa-PBD, usuallyat the initial time point, will also be required (for bleedthroughcorrections as described below). In contrast, when generating images(i.e. to determine the distribution of rac activation) an Alexa-PBDimage is taken at each time point. The reasons for this are discussed inthe ‘Image Processing’ section below. If the cells are to be treatedwith some type of stimulus, it is helpful to take a series of imagesprior to stimulation as controls for noise, bleaching, and otherartifacts.

Image Processing. Image analysis is performed to follow the kinetics oftotal Rac activity within individual cells, displayed as curves ofactivation over time, or to generate images that show the subcellularlocation of rac activation. The proper application of corrections isneeded essential for quantitative imaging. Common image processingoperations can be used for this purpose (Berland et al., in Sluder etal., Video Microscopy at 33 (Academic Press 1998). Procedures for suchimage processing operations will depend on the software package used.The correction factors should be rigorously applied when using the FLAIRsystem, as FRET signals will be low relative to other sources offluorescence in the sample. The FRET signal must purposefully be keptlow, as minimum quantities of fluorescent molecules should be used toprevent perturbation of cell behavior. Hence, FLAIR procedures must bemore carefully controlled than procedures generally used withfluorescent probes.

The following protocols assume use of a cooled CCD camera, whichtypically show low levels of noise, linear response to light intensity,and little variation in response from pixel to pixel. It is valuable touse cameras and software with the greatest possible bit depth (allotmentof computer memory to each pixel to maximize the number of possibleintensity gradations). This is especially important for ratiooperations, which are typically performed using 12 bit images orgreater. Operations are described in the order in which they should beperformed:

1. Registration. For corrections applied in later steps, it is importantthat each of the images taken using different excitation and emissionwavelengths be registered so that the cells lie atop one another, withcell edges and internal features exactly coinciding. Different imageprocessing software will accomplish this in different ways, but manualtranslocation is usually involved so that one image lines up with asecond, fixed image. This is best accomplished by zooming in on cellsand adjusting brightness and contrast to clearly see cell edges andinternal features. Since the GFP-Rac signal is generally the strongest,it was used as the reference image in these experiments. When thebleedthrough corrections described below are performed, errors inregistration often become apparent as ‘shadow effects.’

2. Background subtraction. There are two methods commonly used forbackground subtraction. If the only intention is to follow the changingspatial distribution of the FRET signal over time, and if the background(in the absence of cells) remains uniform across the field of viewthroughout the experiment, then it is sufficient to determine thebackground intensity in several regions of the image outside the cell.The average value of these intensities is then subtracted from eachpixel in the image. This method can also be sufficient for followingqualitative changes in the subcellular location of activation, but itmust be used with caution. Subtle variations in background intensityacross the cell could be large relative to the changes observed in FRET,producing artifacts. When quantifying the kinetics of total cell Racactivity over time (see following sections), it is better to take animage of a region of the coverslip containing no cells or fluorescentdebris, under the same conditions used for taking the real image. Thisbackground image is then subtracted from the real image prior to furtheranalysis. A separate background image must be taken for each type ofimage (GFP, FRET or Alexa) and at each time point when successive imagesare obtained

3. Masking. It is advisable to mask out regions surrounding the cellsprior to further analysis. The edges of the cell are outlined, in mostsoftware either manually or by eliminating all sections of the imagesbelow a certain intensity value (interactive thresholding). The regionsoutside the cell are thus identified and eliminated from furthercalculations. The precise approach for accomplishing this will dependupon the software employed. However, the mask is usually a binary imagewith all values within the cell=1, and all outside=0, and the real imageis multiplied by the mask. The mask is best generated using the GEPimage, which has the strongest signal and therefore the most clearlydefined edges. When determining the total intensity within the cell,analysis should be carried out on the same pixels within the FRET, GFP,and Alexa images. Therefore, the same mask is applied to each imageafter registration, assuring that exactly the same pixels are analyzed.

4. Bleedthrough correction. During FRET it is necessary to excite thedonor fluorophore while monitoring emission from the acceptorfluorophore. It is extremely difficult to design FRET filters that seeonly FRET emission and block all GFP emission, or block all light fromAlexa excited directly rather than by FRET. To correct for“bleedthrough” of such light into the FRET image, the fluorescencefilters must be characterized by taking images of cells containingGFP-Rac or AlexaPBD alone. For example, in bleedthrough correction forGFP, cells are imaged using both the GFP and FRET filter set. Whenobserving the GFP fluorescence through FRET filters, a fixed percentageof the GFP emission will be seen. The total fluorescence intensity isdetermined for both the GFP and FRET images from cells containing onlyGFP-Rac. A GFP bleedthrough factor is computed for each cell by dividingthe intensity through the FRET filters by that through the GFP filters(the ‘bleedthrough factor’ for GFP: FRET intensity/GFP intensity). Thisvalue is plotted against cell intensity for numerous cells, and a lineis fit to this data to produce an accurate value of the bleedthroughfactor. It is important to use background-subtracted images. The processis repeated for Alexa-PBD. When the actual experiment is performed, anAlexa-PBD, GFP-Rac, and FRET image are obtained. After backgroundsubtraction of all three images, the Alexa-PBD and GFP-Rac images aremultiplied by the appropriate bleedthrough factor and subtracted fromthe FRET image. This is an extremely important step that must be appliedcarefully to prevent artifacts that appear to be regions of high FRET,especially as the magnitude of the FRET signal approaches that of thebleedthrough. It is important not to use GFP-Rac or Alexa-PBD imagesthat exceed the dynamic range of the camera (‘overexposure’) as theywill not fully eliminate bleedthrough. Motion artifacts can also produceerrors derived from bleedthrough corrections.

Production of total activation curves (bleaching correction). Todetermine how overall Rac activity within a cell changes over time, thetotal fluorescence intensity within a GFP-Rac and FRET image isdetermined at each time point. The intensity of the FRET image isdivided by that of the GFP-Rac image. This ratio better reflects totalRac activity than does FRET intensity alone. Division by GFP-Racnormalizes out errors due to bleaching of GFP, effects of unevenillumination, and other factors affecting both the GFP and FRET signals.Because all FRET occurs through irradiation of GFP, bleaching of GFPwill decrease both GFP and FRET emissions to the same extent. Therefore,the FRET/GFP ratio will be a measure of Rac activity that is notaffected by bleaching. It is critical that each image be properlybackground subtracted and corrected for bleedthrough. Bleedthroughcorrections are somewhat simplified when generating these curves, asthey need not be carried out on actual images, but simply on the totalintensity values derived from the images. The total intensity of theGFP-Rac image is multiplied by the GFP bleedthrough factor, and theresulting value is subtracted from the FRET intensity. An analogousoperation is performed for Alexa-PBD bleedthrough. One need only obtaina single Alexa-PBD image (usually at the beginning of the time series)and use this for bleedthrough correction of all images in the timeseries. If Alexa-PBD is not irradiated during the experiment, itsbleaching will be negligible and this single image will reflect theactual Alexa-PBD level throughout the entire time series.

Any ratio calculations are best performed using floating pointoperations. Large errors can be generated by software that truncatesnoninteger values into integers to display data as images. Such problemscan be overcome by multiplying the images by a large scalar value priorto division. This value should be as high as possible without causingany pixel to exceed the bit depth of the image file. For example, a12-bit image file can only hold values up to 4095 (2¹²), so the constantselected must not cause the highest intensity value in the image toexceed 4095. When operations are performed on whole cell values ratherthan on images, as with production of the Rac activation curvesdescribed here, it is convenient to determine total cell intensities andthen perform any division operations in a floating point spreadsheetprogram, such as Microsoft Excel.

Examining changes in the localization of Rac activation. The sectionsabove describe how to generate images showing the distribution of FRETwithin cells. A sequence of such images can be compared to show howlocalizations vary with time. While simple examination can suffice toshow distribution changes, the overall intensity of FRET, and hence theperceived activation level, could become successively lower over timedue to bleaching of the GFP. We have found that bleaching is not aserious issue for at least 20 images under the exposure conditionsdescribed here. The total GFP intensity at the beginning and end of theexperiment should be examined to gauge bleaching effects. One cancorrect for bleaching by dividing each pixel in the image by the totalintensity of GFP determined at the same time point.

Many aspects of cytoskeletal control and signaling crosstalk depend uponthe localization of Rho family GTPase activation, and may depend as wellon the level and duration of activation. Signaling control by theprecise dynamics of GTPase activation has been suggested by indirectexperiments, but has been very difficult to quantify or study usingprevious methods. The FLAIR system reported here can reveal Racactivation dynamics in vivo and can accurately report the changingactivation levels within a cell.

EXAMPLE 7 Cellular Localization of Rac GTPase Activation Using p21Activated Kinase (PAK) as a Protein Biosensor

Prevailing evidence suggests that signaling proteins must be tightlyregulated both spatially and temporally in order to generate specificand localized downstream effects. For Rac1 and other small GTPases,binding to GTP is a critical regulatory event that leads to interactionwith downstream targets and regulates subcellular localization. Here weuse FLAIR (fluorescence activation indicator for Rho proteins), toquantify the spatio-temporal dynamics of Rac1 GTP interaction in livingcells. FLAIR reveals precise spatial control of growth factor-inducedRac activation in membrane ruffles, and in a gradient of activation atthe leading edge of motile cells.

Rac is a member of the Ras superfamily of small GTPase proteins (A.Hall, Science 279, 509-14 (1998). It plays a critical role in diversesignaling pathways, including control of cell morphology, actindynamics, transcriptional activation, apoptosis signaling, and othermore specialized functions (L. Kjoller, A. Hall, Exp. Cell Res. 253166-79 (1999). The broad range of events controlled by this GTPaserequires subtle regulation of interactions with multiple downstreamtargets. Accumulating evidence suggests that the effects of Rac are inpart controlled by regulating the subcellular localization of itsactivation through GTP binding. For example, Rac is known to inducelocalized actin rearrangements to generate polarized morphologicalchanges (C. D. Nobes, A. Hall, J. Cell Biol. 144(6), 1235-1244 (1999).GTP exchange factors (GEFs) which regulate nucleotide exchange on RhoGTPases contain a variety of localization domains and may modulatedownstream signaling from Rac (Zhou et al, J. Biol. Chem. 273(27),16782-16786 (1998).

Although control of Rac1 signaling through localized activation isclearly important, it is difficult to study in an intact cell. Here wedevelop and apply a new method based on fluorescence resonance energytransfer (FRET) which can quantify the timing and location of Racactivation through GTP binding. This method is applied to examine thehypothesis that specific and localized Rac1 activation occurs during thecourse of extracellular signal-induced cytoskeletal dynamics.

The design of the Rac nucleotide state biosensor is shown schematicallyin FIG. 5. A fluorescently labeled protein biosensor is introduced intothe cell together with a biologically active GFP-fused Rac (C. Subausteet al, J. Biol. Chem. 275(13), 9725-9733 (2000). This protein biosensoris labeled with an acceptor dye capable of undergoing FRET with GFP.Since the biosensor is derived from a specific GTP-Rac target protein,it binds to GFP-Rac 1 only when the Rac1 is in its activated, GTP-boundform, and produces a localized FRET signal revealing the level andlocation of Rac activation. When cells expressing GFP-Rac were injectedwith the biosensor, we were able to simultaneously map the changinglocation of GFP-Rac1 and the subpopulation of GFP-Rac1 molecules in theactivated, GTP-bound state. FRET is proportional to the amount of GTPbinding, enabling quantitation of changing activation levels. It is alsovery specific, as only biosensor binding to the GFP-tagged protein willgenerate FRET. This approach has the potential to examine many proteinstates, including posttranslational modifications, conformation, andligand binding.

The biosensor was made by fluorescently labeling a domain from p21activated kinase I (PAK1) known to bind selectively to GTP-Rac. PBD(fragment of human PAK1, residues 65-150, with a single cysteine addedin the penultimate N-terminal position) was expressed as a C-terminal6His fusion protein from the pET23 vector (Novagen) and purified from E.coli strain BL21DE3 using Talon metal affinity resin (Clontech) asinstructed by the manufacturer. All GFP constructs were prepared usingthe EGFP mutant (T. Joneson, Mol. Cell Biol. 19(9) 5892-901(1999); T. T.Yang et al., Gene 173, 19-23 (1996)), kindly provided by Dr. Roger Tsienof the University of California, San Diego. GFP-Rac 1 fusion and wildtype Rac1 for in vitro studies were also expressed as 6His constructsand purified using a similar procedure. Purified protein was dialyzedagainst 50 mM sodium phosphate (pH 7.8), and labeled with 7 equivalentsAlexa 546 maleimide (Molecular Probes) at 25 degrees for 2 hours. Theconjugate was purified from unincorporated dye by G25 size exclusionchromatography followed by dialysis. The dye:protein ratio was between0.8 and 1.3, as determined from absorbance of the conjugate at 558 nm(Alexa 546 extinction coefficient 104,000 M⁻¹ cm⁻¹) and 280nm (PBD,extinction coefficient 8,250 M⁻¹ cm⁻¹ plus Alexa absorbance, determinedas 12% of the absorbance at 546). Protein concentration was alsoindependently determined using a Coomassie Plus protein assay (Pierce)and SDS-PAGE calibration with known concentrations of bovine serumalbumin.

The p21 binding domain (PBD, aa 65-150) has been used successfully toprecipitate GTP-Rac 1 from cell lysates (V. Benard, B. P. Bohl, G. M.Bokoch, J. Biol. Chem. 274, 13198-204(1999). In order to produceefficient FRET, the optimum site for attachment of an acceptor dye wasdetermined by analyzing FRET between purified GFP-Rac1 and PBD labeledwith various dyes in different positions. PBD contained no nativecysteines, so the site of labeling could be controlled throughintroduction of a single cysteine, followed by labeling withcysteine-selective iodoacetamide dyes. After considerable optimization,the best candidate was found to be a PBD with cysteine appended to theN-terminus, labeled with commercially available Alexa 546 dye. Thedistance between the Alexa dye at the N-terminus of PBD and thefluorophore in GFP was calculated to be 52 A based on the efficiency ofFRET and assuming random rotation of the fluorophores (Ro=51, n=¼, k²=⅔)(T. Nomanbhoy et al., Biochemistry 35, 4620-4628 (1996). We coined thename FLAIR (fluorescent activation indicator for Rho proteins) for thisnew live cell imaging technique.

FRET between Alexa-PBD and GFP-Rac1 was efficient and dependent onGTP-Rac 1 binding. Using fluorescence excitation wavelengths thatselectively excite GEP (480 nm), fluorescence emission was monitoredwhile maintaining a fixed concentration of GFP-Rac 1 and varyingAlexaPBD concentrations (see FIG. 7 a). Purified GFP-Rac1 (200 nM) wasbound to varying concentrations of GTPYS or GDP at low magnesium by 30min incubation at 30° C. as described previously (U. G. Knaus et al., J.Biol. Chem. 267, 23575-23582 (1992)), using the following nucleotideequilibration buffer: 50 mM Tris HCl (pH 7.6), 50 mM NaCl, 5 mM MgCl₂,10 mM EDTA, and 1 mM DTT. Equal volumes of Alexa-PBD in the same bufferwere added to the GFP-Rac1 solution and fluorescence emission spectra(500-600 nm) were acquired at room temperature and 480 nm excitation.Spectra were corrected for dilution upon Alexa-PBD addition. Alexa-PBDconcentrations were either varied as shown, or maintained at 1micromolar when saturating Alexa-PBD was required. The spectra shownwere corrected for direct excitation of the Alexa fluorophore byacquiring spectra of Alexa-PBD alone at equivalent concentrations, andsubtracting these from spectra shown in FIG. 7. K_(d) were determined byfitting to the equation: Y=A*X/(K_(d)+X). In the FIG. 7, panel A inset,only points actually used in the curve fitting are shown. Higher,saturating concentrations of AlexaPBD were not used because errors fromsubtraction of direct Alexa excitation became larger. Binding ofAlexa-PBD to GFP-Rac resulted in a change in fluorescence intensity ofboth donor (GFP) and acceptor (Alexa) emission. When the GFP and Alexafluorophores were brought into close proximity, FRET caused the Alexa(acceptor) emission to increase while the GFP (donor) emissiondecreased. No change in emission was observed when using eitherunlabeled PBD or Rac1 not fused to GFP, indicating that fluorescencechanges were in fact due to energy transfer (data not shown). BecauseFRET alters donor and acceptor intensities in opposite directions, theratio of emission at these two wavelengths is a sensitive measure of thePBD-Rac1 interaction. The corrected Alexa/GFP emission ratio underwent a4-fold change upon saturation of Rac 1 with GTP (FIG. 7 b). The changein emission ratio versus PBD concentration was fit to the Michaelisequation to derive an apparent K_(d) for PBD-Rac1 binding of 1.1±0.3 μM(FIG. 7 a inset). This is slightly higher than the values that have beendetermined for various unlabeled PAK1 fragments (E. Manser, T. Leung, H.Salihuddin, Z.-S. Zhao, L. Lim, Nature 67, 40-46 (1994); D. A. Leonardet al, Biochemistry 36, 1173-1180 (1997); G. Thompson, D. Owen, P. A.Chalk, P. N. Lowe, Biochemistry 37, 7885-7891 (1998). This may indicatethat the presence of the Alexa may somewhat weaken the bindinginteraction. Our studies in live cells, described below, demonstratedthat this affinity was sufficient for detection of Rac activation invivo, and reversible binding is in fact desirable as it permits thebiosensor to rapidly respond to changes in the location or level of Racactivation. Importantly, Alexa-PBD was shown to minimally perturbRac-GTP binding interactions. The apparent GTPγS dissociation constantwas determined at saturating Alexa-PBD by fitting the experimental datato the Michaelis equation (FIG. 7 b). The derived value of 47±9 nM isconsistent with the previously reported value of 50 nM (L. Menard, E.Tomhave, P. J. Casey, R. J. Uhing. R. Snyderman, J. R. Didsbury, Eur. J.Biochem. 206, 537-546 (1992). This validated application of FLAIR as anindicator of biologically relevant Rac-nucleotide binding.

Much is known about the localized dynamics of actin, but it has beendifficult to explore how signals can generate precisely localized actinbehavior. Rac has been shown to participate in signaling cascadesleading to localized polymerization in several systems, but it isunclear whether Rac activity is constrained to specific subcellularregions, or whether global activation of Rac leads to more localizedactivation of downstream molecules. The precise spatial correlation oflocalized signaling and downstream actin behavior remains completelyunknown. Here, we used FLAIR to study the localization and activation ofRac in two cell systems where actin behaviors are clearly constrained tospecific subcellular regions, the induction of ruffling by growth factorstimulation of quiescent cells, and polarized cell motility induced bywounding.

We examined stimulation of quiescent Swiss 3T3 fibroblasts by serum orplatelet derived growth factor (PDGF) known to initiate membraneruffling and transcription through activation of Rac 1 (Ridley A. J.,Paterson H. F., Johnston C. L., Diekmann D., Hall A., Cell 70,401-10(1992); P. T. Hawkins, Curr. Biol. 5(4), 393-403 (1995)). For serumstimulation experiments, Swiss 3T3 fibroblasts (ATCC, passage 15-27)were plated on glass cover slips and then maintained in Dulbeccosmodified Eagle's medium (DMEM) with 10% fetal calf serum (FCS), 1%L-glutamine and 1% penicillin-streptomycin for at least 24 hours. Mediawas then replaced with media containing only 0.5% FCS, and cells weremaintained for 42 hours. Cells were transfected by microinjecting 200micrograms/ml GFP-Rac1 c-DNA into cell nuclei 2-8 hours prior to theexperiment. The EGFP mutant was used in all experiments, cloned andexpressed as previously described (C. Subauste et al, J. Biol. Chem.275(13), 9725-9733 (2000)). Cells expressing the GFP-Rac were thenmicroinjected with 100 micromolar Alexa-PBD, mounted in a heated chamberon a Zeiss axiovert 100 TV microscope and maintained in Dulbecco'sphosphate buffered saline (DPBS) (GIBCO) to reduce backgroundfluorescence. Cells were then stimulated by replacing the media withDPBS containing 10% FCS or 50 ng/mL PDGF. Images were obtained every 30seconds using a Photometrics PXL cooled CCD camera with 1×1 or 3×3binning, and a Zeiss 40×1.3 NA oil immersion objective. Fluorescencefilters from Chroma were as follows: GFP: HQ480/40, HQ535/50, Q505LP;FRET: D480/30, HQ610/75, 505LP; Alexa:HQ 545/30, HQ 610/75, Q565LP.Cells were illuminated using a 100 W Hg arc lamp. Exposure times for 3×3binning were: GFP-0.1 seconds, Alexa-PBD-0.1 seconds, FRET-0.5 seconds.For 1×1 binning, GFP-1 second, Alexa-PBD-1 second, FRET-5 seconds.

Levels of exogenous proteins had to be limited to concentrations thatwould not perturb cell behavior. We determined the intracellular levelsof Alexa-PBD and GEP-Rac 1 that altered normal serum-induced ruffleformation (FIG. 8), and kept exogenous protein below these levelsthroughout our studies.

Inage triplets of GFP, FRET, and Alexa fluorescence were taken at eachsuccessive time point before and after stimulation. Thus, as shown inFIG. 9, both the changing localizations of GFP-Rac, and the level andlocation of Rac activation could be monitored. Images were firstbackground subtracted and carefully registered to ensure accurate pixelalignment. The GFP-Rac image was then thresholded, changing theintensities of all pixels outside of the cell to zero. Thresholding wasbased on the GFP image since it had the largest signal to noise ratio,providing the clearest distinction between the cell and background. Thethresholded GFP-Rac image was used to generate a binary image with allvalues within the cell=1 and all outside=0. The FRET and Alexa-PBDimages were multiplied by the binary image, assuring that exactly thesame pixels were analyzed in all three images. Emission appearing in theFRET image from direct excitation of Alexa and GFP was removed bysubtracting a fraction of the GEP-Rac and Alexa-PBD images from the FRETimage. This fraction depended on the filter set and exposure conditionsused. It was determined, as described in detail elsewhere (C. E.Chamberlain, V. Kraynov, K. M. Hahn, Methods Enzymology (In Press2000)), by taking images of cells containing only GFP-Rac or Alexa-PBDalone, and quantifying the relative intensity of emission in the FRETchannel and that in the GFP or Alexa-PBD channel. A broad range ofintensities was examined and a line was fit to these for accuratedeterminations. These corrections had to be applied carefully whenstudying rapidly moving objects such as ruffles. If the ruffle movedbetween acquisition of the FRET, GFP, or Alexa images, the subtractivecorrection process would remove light from the FRET image in the wrongplace, generating artefactual FRET localizations. Data from movingfeatures was used only when careful inspection showed the feature to becoincident in the Alexa, GFP, and FRET images, and controls wereperformed with images taken in different orders. A low pass filterkernel was applied to the corrected FRET image to remove high frequencynoise (K. Castleman, Digital Image Processing (Prentice Hall, NewJersey, 1996), pp. 207-209). Image processing and microscope automationwere performed using Inovision ISEE software. Images were contraststretched and formatted for display using Adobe Photoshop software.

We tested Rac and PBD fused to GFP mutants that undergo FRET (ECFP andEYFP) (S. Dharmawardhane, D. Brownson, M. Lennartz, G. M. Bokoch, J.Leukoc. Biol. 66(3), p. 521-527 (1999); B. A. Pollok and R. Heim, TrendsCell Biol. 9(2), 57-60 (1999)). Unfortunately, their spectral overlapwas more problematic than that of Alexa and GFP, making the correctionsdescribed here more difficult In addition, the GFP mutants showedroughly 25% the FRET of FLAIR. We decided to use FLAIR for thesereasons, but the ability to monitor Rac activity simply through proteinexpression may justify using GFP mutants in some applications.

Simple GFP-Rac fluorescence revealed pools of Rac1 at the nucleus, inthe juxtanuclear region, and in small foci throughout the cell prior tostimulation. Confocal and deconvolution imaging showed the nuclear Racto be concentrated at the nuclear envelope, and expression andimmunostaining of HA-tagged Rac1 indicated that these localizations werenot an artifact of GFP tagging. Addition of PDGF or serum led toformation of moving ruffles throughout the cell periphery within 2minutes. These contained GFP-Rac, shown by phalloidin staining of fixedspecimens to colocalize with actin (data not shown). The FRET imagesshowed a stark contrast between the level of Rac activation in theruffles and the nucleus. No FRET was seen at the nucleus despite thehigh concentration of Rac1 there, while the moving ruffles showed thehighest FRET, clearly restricted to the ruffle and discernable from therest of the cell. The Rac activation remained tightly correlated withthe position of the ruffle even as it moved throughout the cell.

As a negative control, FRET was imaged in cells expressing a mutant ofGFP-Rho, a close relative of Rac that should not bind PBD. This GFP-RhoQ63L mutant, which generates high levels of GTP-bound protein, producedclear Rho localizations but no corresponding FRET signals (data notshown).

These studies provide the first direct evidence that Rac1 activation isrestricted to the site of actin polymerization, independent of theoverall distribution of the protein. Rac activation was tightlycorrelated with moving ruffles, indicating that structures specificallyassociated with the ruffle were either binding and concentratingactivated Rac or that growth-factor induced Rac activation wasspecifically localized to ruffles. The function of the Rac found at thenuclear envelope remains uncertain. It may be activated for regulationof transcription at times later than those tested here, or may beactivated for an unknown role by other stimuli. When activation wasconcentrated in a small area such as a ruffle, spatially resolved FRETcould detect significant activation changes too small to appreciablyalter the overall levels of cellular Rac activity. Our data showed thatFRET provided much greater sensitivity and selectivity than followingRac activation simply by imaging Alexa-PBD localization (FIG. 9, panelsC and D). FRET produces much lower backgrounds and provides completeselectivity even when the biosensor can bind to multiple proteins (i.e.Alexa-PBD also binds to cdc42). Unlike simple localization, FRET canprovide quantitative measures of activation levels. It is important tonote that the Alexa-PBD could have been sterically hindered fromreaching Rac1 in some locations, so that not all activated Rac1 may havebeen detected. Nonetheless, a FRET signal in a given location doesreveal that Rac activation is occurring there.

Rac has been shown to be essential for the directed movement of cellsduring chemotaxis, and for extension of the front end of cells duringmotility (C. Y. Chung et al, Proc. Natl. Acad. Sci. U.S.A. 97(10),5225-5230 (2000). We used FLAIR to ask if Rac activation in polarized,motile cells occurred in particular subcellular localizations toregulate localized actin behaviors. A ‘wound’ was scraped in a monolayerof confluent Swiss 3T3 fibroblasts, causing cells to become polarizedand move into the open space. For wound healing experiments, Swiss 3T3fibroblasts were induced to undergo polarized movement as previouslydescribed (R. DeBiasio, G. R. Bright, L. A. Ernst, A. S. Waggoner, D. L.Taylor, J. Cell. Biol. 105, 1613-22 (1987). The cells were cultured inDulbecco's modified Eagle's medium (GIBCO) supplemented with 10% fetalcalf serum at 37° C. Cells were trypsinized and then plated on glasscoverslips. They were grown to a confluent monolayer and maintained foran additional 3-4 days. Cells were then wounded by creating a straightlaceration with a sterile razor blade. Cells along the edge of the woundwere microinjected with 200 micrograms/ml GFP-Rac c-DNA. Six hours afterthe wound was formed, cells expressing the GFP-Rac were microinjectedwith 100 micromolar Alexa-PBD and allowed approximately 10 minutes forrecovery. Media was then replaced with DPBS containing 10% FCS to reducebackground fluorescence. Images were obtained as described above, usingexposure times of 1 second for GFP, 1 second for Alexa-PBD, and 5seconds for FRET. FLAIR revealed highest Rac activation in thejuxtanuclear area, and a gradient of Rac activity highest near theleading edge and tapering off towards the nucleus (FIG. 10).

Quantitative analysis clearly indicated that the gradient was correlatedwith the direction of movement. In individual cells, total Rac activitywas measured in two subcellular locations: where activity was highest atthe front of the cell, and lowest at the very rear of the cell. Thesevalues, measured in squares three microns on a side, were used tocalculate the gradient (Percent gradient=100×[front−back]/back). Of the16 cells examined, twelve had higher Rac activity at the leading edge,while four showed a slight negative gradient. For cells with high Racactivity at the leading edge, the gradient was 128±51%, while for cellsshowing the reverse gradient, it was only 9±4% (mean±standard error).The gradient was much broader than the narrow area at the leading edgewhere actin polymerization occurs (Y. L. Wang et al, J. Cell Biol 101,597-602 (1985; J. A. Theriot and T. J. Mitchison, Nature 352, 126-131(1991). Other activities required for motility, such as depolymerizationof fiber networks to recycle monomers and delivery of molecules to theleading edge (O. D. Weiner et al., Nat. Cell. Biol. 1, 75-81 (1999)occur throughout the region where Rac is activated. Perhaps Rac isacting over a broader cell area to activate multiple downstreameffectors, each producing different effects in more restrictedlocations. Other studies have shown tight localization of moleculesdownstream of Rac, at the leading edge or in regions immediately behindit to regulate a variety of functions associated with motility (F.Michiels et al., Nature 375, 338-340 (1995).

The prevalence of Rac activation around the nucleus was quantified byscoring 16 cells. All cells showed both juxtanuclear and nuclear GFPfluorescence. Of these, fourteen showed juxtanuclear FRET, and noneshowed nuclear FRET. It was noteworthy that small areas of the nucleussometimes showed a FRET signal, but these could be due either tocytoplasmic Rac associated with the nuclear envelope or to juxtanuclearlocalizations lying over the nucleus. The localization of activationwithin the juxtanuclear Rac often did not parallel Rac distribution,with “hot spots” of FRET within areas of lower Rac concentration. Themeaning of the juxtanuclear Rac localizations is unclear, but theirmorphology and distribution suggests activation within the ER, golgi. orvesicle populations, perhaps consistent with recent reports suggestingan important role for Rac in ER to golgi transport (M. P. Quinlan, CellGrowth Diff. 10(12), 839-854 (1999), and in pinocytic vesicle cycling(Ridley A. J., Paterson H. F., Johnston C. L., Diekmann D., Hall A.,Cell 70,401-10 (1992).

In summary, we have described a novel approach to quantify the spatialdistribution and rapidly changing levels of Rac signaling in livingcells. FLAIR provided the first direct demonstration that Rac activityis spatially regulated to generate specific actin behaviors in differentsubeellular regions. Activated Rac was tightly coupled to small membraneruffles even as the ruffles moved throughout the cytoplasm, yet wasbroadly distributed as a gradient at the leading edge of motile cells.These very different activation patterns suggest that the cell willutilize different distributions of activated Rac depending on the numberand localization of downstream targets.

Ultimately, FLAIR can reveal how different stimuli interact to affectRac through the complex circuitry of an intact cell. We have focusedhere on spatial control of signaling. The ability of the biosensor toquantify the level and kinetics of activation should also prove veryuseful, as accumulating evidence indicates that Rac and related proteinsdo not function as simple binary switches. Different levels and kineticsof activation produce profoundly different results (T. Joneson, Mol.Cell Biol. 19(9) 5892-901(1999). Using FLAIR together with otherbiosensors of different wavelengths it should be possible to examine thebalance between Rac, Ras, and other protein activation levels undergoingrapid changes in real time. With increasing access to FRET imagingequipment, the technique we describe provides a relativelystraightforward way to greatly extend the utility of readily accessibleGFP fusion proteins.

EXAMPLE 8 Activation Biosensors for cdc42 and Erk2

Like Rac, cdc42 is a member of the Rho family of small GTPases involvedin signal transduction in eukaryotic cells. Cdc42 becomes “activated” byreleasing GDP and binding GTP. Such GTPases interact with a host ofdownstream effectors, ultimately resulting in one or the other cellularresponse via a variety of phosphorylation cascades. In theseexperiments, a unique synthetic fluorophore with environment-sensitivefluorescence properties was linked to the cdc42 binding domain (“CBD”)of the Wiscott-Aldrich Syndrome Protein (WASP) to generate a CBDbiosensor. Upon binding to the cdc42, this fluorescent CBD biosensor isable to increase its fluorescence intensity by up to 3.5-fold, providinga convenient measure of endogenous cdc42 activation in living cells orfor in vitro applications (concept outlined in FIG. 6 b).

Mitogen-activated protein kinase (MAP kinase) was first identified as aprotein phosphorylase which is activated when a growth factor is addedto cultured cells (Proc. Natl. Acad. Sci. USA, 84, 1502-1506, 1987).However, subsequent research revealed that this enzyme is involved invarious vital phenomena such as neuronal differentiation (J. Biol.Chem., 265, 4730-4735, 1990), activation of immune cells (J. Immunol.,144, 2683-2689, 1990) and secretions (J. Cell Biol., 110, 731-742,1990). MAP kinase is also called Extracellular Signal-Regulated Kinase,or ERK. Cloning of the gene for human ERK gene showed that it consistsof several molecular species with high homology, but the main speciesare two, namely ERK1 and ERK2. These two proteins are highly homologous(84.7%) (FEBS LETT., 304, 170-178, 1992). Mitogen-Activated ProteinKinase Kinase (called MEK) can interact with and stimulate ERK2.

Experimental Protocols:

Production of recombinant proteins. DNA encoding the Cdc42-bindingfragment of human WASP containing the CRIB motif and surrounding aminoacids (WASP amino acids 201 to 321) was amplified by PCR from ATCC clone#99534. This peptide fragment has the following amino acid sequence (SEQID NO:13 ). DIQNPDITSSRYRGLPAPGPSPADKKRSGKKKISKADIGAPSGFKHVSHVGWDPQNGFDVNNLDPDLRSLFSRAGISEAQLTDAETSKLIYDFIEDQGGL EAVRQEMRRQEPLPPPPPPS

The full sequence of the WASP protein is as follows (SEQ ID NO:14):MSGGPMGGRP GGRGAPAVQQ NIPSTLLQDH ENQRLFEMLG RKCLTLATAV VQLYLALPPGAEHWTKEHCG AVCFVKDNPQ KSYFIRLYGL QAGRLLWEQE LYSQLVYSTP TPFFHTFAGDDCQAGLNFAD EDEAQAFRAL VQEKIQKRNQ RQSGDRRQLP PPPTPANEER RGGLPPLPLHPGGDQGGPPV GPLSLGLATV DIQNPDITSS RYRGLPAPGP SPADKKRSGK KKISKADIGAPSGFKHVSHV GWDPQNGFDV NNLDPDLRSL FSRAGISEAQ LTDAETSKLI YDFIEDQGGLEAVRQEMRRQ EPLPPPPPPS RGGNQLPRPP IVGGNKGRSG PLPPVPLGIA PPPPTPRGPPPPGRGGPPPP PPPATGRSGP LPPPPPGAGG PPMPPPPPPP PPPPSSGNGP APPPLPPALVPAGGLAPGGG RGALLDQIRQ GIQLNKTPGA PESSALQPPP QSSEGLVGAL MHVMQKRSRAIHSSDEGEDQ AGDEDEDDEW DDThe DNA fragment encoding SEQ ID NO:13 was subcloned into pET23a(Novagen) as a C-terminal 6His fusion. Site-specific cysteine mutantswere constructed by QuikChange (Stratagene) mutagenesis using syntheticoligos and the presence of mutations was confirmed by DNA sequencing.Resultant constructs were transformed into BL21DE3 strain of E. coli(Novagen), and the proteins were produced by expression at 30° C. for 5hours in 1 L Leuria-Bertani media (Sigma) in the presence of 100 μg/mlof carbenicilin. Expression was induced with 0.5 mM IPTG at OD₆₀₀=0.8-1.Cells were collected by centrifugation and stored at −20° C. until use.

Cell pellet was resuspended in cold lysis buffer (25 mM Tris-HCl, pH7.9, 150 mM NaCl, 5 mM MgCl₂, 5% glycerol, 1 mM PMSF, 2 mMβ-mercaptoethanol), and briefly sonicated on ice. Lysozyme and DNasewere added to the suspension to a final concentration of 0.1 mg/ml and100 U/ml, respectively, and solution was incubated with occasionalstirring at 4° C. for 30 min. Lysate was centrifuged (12,000 g, 30 min),and the clarified supernatant was incubated with 1 ml of Talon resin(Clontech) at 25° C. for 30 min. The resin containing bound CBD wasseparated from the lysate by brief low-speed centrifugation and washedtwice with 15 ml of lysis buffer. Finally, the resin was washed with thelysis buffer, supplemented with 10 mM imidazole, and poured into acolumn. Elution was performed with 5 ml of the lysis buffer, containing60 mM imidazole. Fractions containing bulk of CBD (as evidenced by SDSgel) were combined and dialyzed against 1 L of dialysis buffer (25 mMNa₂HPO₄ (pH 7.5), 10 mM NaCl) for 5 hours at 4 C. Solution was thenconcentrated using Aquacide powder to a final protein concentration of 2to 10 mg/ml, and dialyzed once again against dialysis buffer. Finalpreparation was flash-frozen in 100 μL aliquots and stored at −80° C.Generally, 1 to 3 mg of CBD was obtained from 1 L of cell culture.Recombinant 6His-tagged cdc42, RhoA, Rac1, ERK2 and MEK were produced byanalogous procedures. The enzymes were determined to be >90% active bythe GTP binding assay (Knaus, U. G., Heyworth, P. G., Kinsella, B. T.,Cumutte, J. T., and Bokoch, G. M. (1992) J. Biol. Chem. 267,23575-23582).

Conjugation of CBD with fluorescent dyes—Dialyzed CBD samples (100-150μM) were gently inverted with 6 to 7-fold molar excess of the reactivedye at 25° C. for 3 to 4 hours. The reaction was stopped by addition of10 mM dithiothreitol (DTT), and the mixture was incubated for 15 min.Unreacted dye was separated from the labeled protein using G25-Sepharose(Pharmacia) gel filtration column equilibrated and developed with 25 mMNa₂HPO₄ (pH 7.5). Purity of the eluting fractions was analyzed byrunning an aliquot on an SDS gel and visualizing the fluorescence. Onlythe fractions containing minimal amounts of free dye were used in thesubsequent experiments. Dye-to-protein ratio was determined by measuringCBD concentration (ε²⁸⁰=8,250 M⁻¹), and A4C concentration at 617 nm(ε=70,000 M⁻¹ in dimethylsulfoxide) or Alexa546 at 554 nm (ε=104,000 M⁻¹in 50 mM potassium phosphate, pH 7.0). Concentrations of CBD wereindependently confirmed by Coomassie Plus assay (Pierce) calibrated withbovine serum albumin as a standard. Dye-to-protein ratios thus obtainedvaried between 0.8 and 1.2, 1.7 and 2.1 for the single-dye and dual-dyeconjugates, respectively. Aliquots of the labeled CBD (15 to 50 μM) werestored at −80° C. No significant loss of binding ability was observedafter 6 months of storage. In this example, CBD-conjugates were madewith the dye shown in FIG. 11, and are referred to as mero-CBD (formerocyanines dye conjugated to CBD).

Dye Attachment Single Site on ERK2—The ERK2-dye adduct was producedusing conditions which should label only the single solvent-exposedcysteine on ERK. This exposed cysteine was located away from knownprotein interaction sites. A maleimide reactive group on the dye wasattached to this cysteine residue at pH 7.5. The attachment of label andthe activation of ERK2 was examined under different conditions (FIG.23). ERK2 was activated with MEK for 0-60 min, as indicated in FIG. 23.When Mg and ATP were added, ERK2 was phosphorylated; no suchphosphorylation was observed when no Mg, ATP or MEK was present (FIG.23).

Fluorescence increase as a measure of ERK2-MEK interaction—Thefunctionality and specificity of ERK2 biosensor was characterized bymeasuring fluorescence in the presence and absence of saturating amountsof ATP (FIGS. 24 and 25). An approximate 1.6-fold increase in emissionwas observed, when the Erk2 was phosphorylated (FIG. 24). No effect onErk2 fluorescence was observed when no MEK was present (FIG. 25). Thisresult demonstrated the suitability of the present methods for detectingactivation of ERK2 in live cells.

Analyses of the solvatochromic activation indicator—A solution ofmero-CBD (300 nM) in assay buffer was mixed 1:1 (v/v) with solutions ofcdc42 (concentration as indicated), pre-equilibrated with 10 mM GDP orGTPγS as described in Knaus, U. G., Heyworth, P. G., Kinsella, B. T.,Cumutte, J. T., and Bokoch, G. M. (1992) J. Biol. Chem. 267,23575-23582. Emission wavelength of 630 nm and excitation wavelength of600 nm were used to acquire excitation and emission spectra,respectively. For nucleotide dependence, cdc42 (500 nM) waspre-incubated with varying concentrations of GTPγS (1 to 500 nM). Rac1and RhoA GTPases were pre-equilibrated with GTPγS in the same manner.

Assay of cdc42 activity in stimulated neutrophil cell lysates.—Freshlyprepared neutrophils (2.5×10⁷ cells/sample) were incubated in aKrebbs-Ringer HEPES buffer supplemented with 5.5 mM glucose (KHRG), inthe presence of 1 mM CaCl₂ and 1 μM of fMet-Leu-Phe tripeptide (FMLP)for various periods of time at 37° C. (see Benard, V., Bohl, B. P., andBokoch, G. M. (1999) J. Biol. Chem. 274, 13198-13204). Stimulation wasstopped by adding equal volume of 2× lysis buffer, supplemented with 2%Nonidet P-40, 2 μg/ml aprotinin. After a brief vortexing the lysateswere clarified by centrifugation and immediately analyzed for cdc42activity with mero-CBD as described above. In control samples, thelysates were pre-equilibrated with either GDP or GTPγS at 30° C. for 20to 30 min. This was a convenient method to determine the extent ofcdc-42 activation in cell lysates, as shown in FIG. 16.

Using the structural information available (Abdul-Manan et al. (1999)Nature 399, 379-383; Kim et al. (2000) Nature 404, 151-158), we hadselected three positions for placing the fluorescent dye within the CBDpeptide (1233, D264 and F271, by WASP numbering), that could experiencea considerable change in solvent polarity as a result of binding tocdc42 (see FIG. 14). F271 and D264 are located in the loop contactingthe effector “switch” domain of the GTPase, whereas I233 appears tointeract with the hydrophobic pocket formed by residues at theN-terminus of cdc42 (see FIG. 14). The three CBD residues were mutatedto cysteines, easily amenable to covalent modification with thethiol-reactive derivatives of the solvatochromic dyes. Recombinantmutant CBD proteins were overexpressed in bacteria, purified andsite-specifically modified with several of solvatochromic dyes. Only theconjugates with the dye shown in FIG. 11 are described here.

Among the three mutants tested, the mero-CBD-F271 C conjugate exhibitedthe largest (ca 3.5-fold) fluorescence change in response to binding ofactivated cdc42 (see FIG. 15).

Fluorescence increase as a measure of CBD-cdc42 interaction—Thefunctionality and specificity of mero-CBD was characterized by measuringfluorescence in the presence of saturating amounts of cdc42-GDP orcdc42-GTPγS. Approximately 3.5-fold increase in both excitation andemission maxima was observed, when the probe was bound to cdc42-GTPγS,but not cdc42-GDP. Negligible increase (<5%) was also observed in thepresence of activated Rac1. No effect on the mero-CBD fluorescence wasobserved when RhoA-GTPγS was present. Furthermore, even in the presenceof excess of activated Rac1 and RhoA, GDP- and GTPγS-bound forms ofcdc42 were easily distinguished by CBD-A4C fluorescence (data notshown). This result demonstrated the suitability of the CBD bisensor foruse in live cells where different Rho GTPases may be present atcomparable concentrations.

FIG. 17 demonstrates use of the biosensor in living cells. To eliminatepotential artifacts due to varying cell thickness, uneven illumination,etc., the biosensor was loaded into the cell together with CBD labeledwith nonresponsive Alexa546 fluorophore. The ratio of the mero-CBD imageto the CBD-Alexa image provided a quantitative measure of the extent ofGTPase activation. The lighter, warmer colors show areas of higher cdc42activation.

EXAMPLE 9 Rhodamine-Tagged Peptide Biosensors

Proteins within living cells were fluorescently labeled in situ, withoutisolation or reintroduction of the protein. A short peptide tag derivedfrom the leucine zipper of GCN4 transcription factor was fused to acellular protein and this fusion protein was expressed in live cells. Asecond peptide from GCN4, which binds with high affinity to the peptidefused onto the cellular protein, was covalently labeled with rhodamineand also introduced into live cells, where it specifically andselectively labeled the tagged protein. Attachment of rhodamine at thechosen site provided good peptide-peptide binding affinity (5 nM Kd).Two proteins were labeled and visualized in living cells, thecytoplasmic protein α-actinin, and a protein spanning the endoplasmicreticulum (ER) membrane, the α-chain of the high affinity IgE receptor(F_(c)εRI). The latter membrane bound protein has previously beeninaccessible to synthetic dye attachment through isolation and labeling.

Materials and Methods

Peptide synthesis. Peptides were synthesized manually using solid-phasemethods with in situ neutralization and HBTU activation procedures forBoc chemistry, on either —OCH2-Pam or MBHA resins as previouslydescribed. Standard Boc-protecting groups strategies were employedexcept for Lys 21 on the labeled peptide, which was protected with anFMOC base-labile protecting group to allow for selective deprotection.Purification was performed using reversed-phase high performance liquidchromatography (RP—HPLC) to obtain peptides greater than 95% pure byanalytical HPLC (UV detection at 214 nm and linear gradients of solventB (0.09% TFA in 90% acetonitrile/10% water) in solvent A (0.1% TFA inwater)). Labeled peptide expected mass (w/o FMOC)=3846, observedmass=3846±0.7. Protein tag peptide expected mass=3735, observedmass=3735±0.7.

Derivatization of the Labeled Peptide at Lys 21 with Rhodamine.

The labeled peptide employed had the following sequence. (SEQ ID NO:15)CEMAQLEKEVQALESEVASLEKEVQALEKEVAQR-NH₂

This peptide binds with specificity to the following peptide (“tag”)sequence. (SEQ ID NO:16) KMAQLKKKVQALKSKVASLKKKVQALKKKVAQR-NH₂Lysine 21 of the peptide having SEQ ID NO:15 was selected as the sitefor tetramethylrhodamine attachment. During Boc-solid phase synthesis,the lysine was protected with a fluorenylmethyloxy-carbonyl protectinggroup, enabling selective deprotection and ready labeling while thepeptide was still attached to the synthesis resin. Peptides containingthree heptamer repeats were used to produce low nanomolar affinities.

To begin the attachment procedure, 20 mg N-hydroxysuccinimide (0.174mmol) and 30.6 mg of 5-(and 6-) tetramethylrhodamine (0.071 mmol) weredissolved in 300 microliters of DMF. To this mixture, 10.85 microlitersof diisopropylcarbodiimide were added. The reaction was then mixed andincubated at room temperature for 2 hours. After 2-3 minutes,diisopropylurea precipitation was evident in the activated dye reaction.200 mg of resin with attached peptide (˜0.05 mmol peptide) was treated 3times with 50% piperidine in DMF to selectively remove the FMOCprotection of Lys 21 (total piperidine deprotecton time was 15 minutes).Deprotection of Lys 21 was confirmed by quantitative ninhydrin assay.After FMOC removal, the resin was thoroughly washed with DMF and drainedcompletely. The in situ activated tetramethylrhodamineN-hydroxysuccinimide ester was diluted to 400 microliters and added tothe peptide-bearing resin dropwise until the resin was saturated withactivated dye solution. The reaction was incubated at room temperaturefor 4 hours, washed with DMF and then with DCM/MeOH (1/1) until washescontained minimal color (4-5 washes each). The resin was dried undervacuum overnight. Standard protocols for cleavage/deprotection andpurification of tetramethylrhodamine-labeled label peptide wereutilized. Expected mass=4258, Observed mass=4258±0.36.

Peptide in vitro interaction measurements. Steady-state fluorescencemeasurements were performed on an SLM 8100 spectro-fluorometer at 25° C.with spectral bandwidths set to 8/16/8 nm and 16/16 nm for excitationand emission, respectively. The absorption of the tetramethylrhodamine(TMR) dye linked to the lysine side-chain was used to calculate thesolution concentration of the peptide in 10 fold diluted PBS, using theassumption that the extinction coefficient of lysine-linked TMR is thesame as that of free dye. The TMR absorption maximum was 553 nm on thepeptide vs. 550 for free dye. The extinction coefficient of the free dyewas 75916 M⁻¹ cm⁻. For these assays only, the sequence YGRKKRRNRRRP (SEQID NO:17) was appended to the N-terminus of the rhodamine-containingpeptide, as this sequence was being tested in a parallel study of cellimport. For the anisotropy titrations, the labeled peptide at aconcentration of 12.52 nM was incubated with a stock solution ofunlabeled peptide in 0.1× PBS. Titrations were performed at magic anglesetting with the excitation wavelength set to 553 nm and the emissionwavelength to 580 nm. The spectra were corrected for Raman scatteringfrom the PBS solution, for dilution, and checked for interactions of thelabel with the ligand by a parallel titration with label peptide using5-carboxytetramethylrhodamine in PBS in the reference cuvette. For eachaddition of unlabeled peptide, ten anisotropy measurements were taken,and the total intensity and anisotropy were calculated according tostandard equations with the G-Factor consistency determined at eachtitration point. The average of the anisotropy was used for dataanalysis. Standard deviations were <1%. If the quantum yield of thefluorophore changed upon binding, this change had to be taken intoaccount for calculation of the ratio between free and boundbiosensor-tag. The anisotropy binding data were therefore fit to thefollowing equation that describes the anisotropy enhancement oftetramethylrhodamine in the labeled peptide (TMRPep) as function of thetotal concentration of added unlabeled peptide (UPep).$r = \frac{r_{\min} + \frac{\left( {{r_{\max}*Q} - r_{\min}} \right)*\left\lbrack {{TMRPep}/{UPep}} \right\rbrack}{\left\lbrack {({TMRPep})\bullet} \right\rbrack}}{\frac{1 - {\left( {1 - Q} \right)*\left\lbrack {{TMRPep}/{UPep}} \right\rbrack}}{\left\lbrack {({TMRPep})\bullet} \right\rbrack}}$with Q=q_(bound)/q_(free), the ratio between quantum yield of TMRPep inbound to free form, and[TMRPep/UPep]=(TMRPep]+[UPep]+Kd]−(([TMRPep]+[UPep]+Kd])²−4*[TMRPep]₀*[UPep]₀^(1/2))/2*[TMRPep]₀.Expression Constructs

The various constructs used in this study were similarly prepared usingstandard cloning (Sambrook et al) of PCR-generated coding sequences intothe indicated vectors. The sequence of all constructs was confirmedusing an ABI model 377 version 3.0 DNA sequencer.

Cloning pEGFP-α-actinin1-peptide tag. The oligonucleotides,5′GAGCTGTACAAGGGAGG-CATGAAGATGGCCCAGCTG AAG 3′ (SEQ ID NO:18) and5′GTCGCGGCCGCCTTATCGCTGGGCCAC-CTTCTTC3′ (SEQ ID NO:19) synthesized byGibco Life Technologies, were used to amplify the peptide tag insert.The humanized DNA sequence for the peptide tag, derived from theavailable peptide, was amplified from pCDNA-Nova2-EGFP-cdc42 (T17N) toincorporate a BsrGI restriction site on the 5′ end and a NotIrestriction site on the 3′ end. These blunt end PCR products produced bypfu polymerase were ligated into pEGFP-N1-α-actinin. After BsrGI andNotI restriction enzymes were used the construct was gel purified(Qiagen Qiaquick Gel Extraction Kit) and ligated with T4 ligase (Gibco,Life Technologies) into the pEGFP-N1-αactinin1 vector downstream ofEGFP. The pEGFP-N1-α-actinin1-Nova2 sequence was verified throughsequencing performed by The Scripps Research Institute core facility.

Cloning of peptide-tagged FcεRI α-chain. The cDNA encoding the humanhigh affinity IgE receptor (FcεRI) α-chain containing the NOVA-2 tagpositioned at C-terminus was generated by a succession of four PCRreactions to sequentially build the desired full length coding sequence,which was then cloned into the pcDNA3.1(+)zeo vector (Invitrogen,Carlsbad, Calif.) to sequentially build the desired full length codingsequence. Each amplification used the 5′ primer:5′-GACTGGATCCGAGTCCATGAAGAAGATGGCTCCTGCC (SEQ ID NO:20) together withthe 3′ primers described below, using human FcεRI α-chain plasmid astemplate and Pfu polymerase (Stratagene, San Diego, Calif.) with 25-30cycles of 94° C. for 1 min; 65° C. for 1 min; and 72° C. for 2 min,followed by one cycle of 72° C. for 10 min. The following downstream PCRprimers were used to generate PCR products 1-4, respectively. (SEQ IDNO:21) 1:5′CTGAACTTTCTTCTTGAGCTGGGCCATCTTACTTCCGCCACCGTTG  TTTTTGGGGTTTGGCTTAGG; (SEQ ID NO:22)2:5′CACCTTTTTCTTCAAGCTCGCCACTTTGCTCTTGAGCGCCTGAACT   TTCTTCTTGAGCTGGGC;(SEQ ID NO:23) 3:5′GGCCTCGAGTCAACGTTGAGCGACTTTCTTTTTTAAGGCTTGCACC  TTTTTCTTCAAGCTCGCCAC; (SEQ ID NO:24)4:5′-CTCGATCGCTCGAGTCAACGTTGAGCGACTTTCTT.The full-length PCR product from reaction 4 was isolated and then clonedas a Xho I/Hind 3 fragment into the pcDNA3.(+) zeo vector (Sambrook, J.,E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A LaboratoryManual, 2nd ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).The nucleotide sequence of the NOVA2-tagged α-chain coding sequence wasconfirmed on an ABI model 377 version 3.0 DNA sequencer.Cloning of the GFP-MHC HLA B27 Fusion Protein.

The GFP protein was fused to the ER-resident major histocompatabilitycomplex I glycoprotein D_(b) (MHC). The cDNA encoding the human HLA B27was generated by PCR amplification using the following oligonucleotideprimers: 5′-GGGGATCCTCTCAGACGCCG-3′ (SEQ ID NO:25) and5′-CATGCCATGGCTCCGGATCCACCAGCTGTGAGAGACAC-3′ (SEQ ID NO:26) to produceBamHI and NcoI ends. This product was ligated with the GFP codingsequence that had been previously excised from the pSGFP vector as aNcoI-NotI fragment. This ligation product was then cloned the into theBamHI-NotI sites of the vector pcDNA3 to produce the desired MHC-GFPfusion protein construct.

Cell culture and imaging. Cos-7 cells were cultured and transfected withFuGENE6 (Roche Molecular Biochemicals) according to the manufacturer'sprotocol. Cells expressing EGFP-α-actinin were microinjected with 20-μMRhodamine-labeled peptide in water. After injection, cells were washedwith Dulbecco's phosphate buffered saline (DPBS) (Life Technologies)with 10% FBS, then mounted in a heated chamber on a Zeiss Axiovert 100TV microscope and maintained in DPBS with 10% Fetal Bovine Serum (FBS)to reduce background fluorescence. Images were obtained using aPhotometrics PXL cooled CCD camera with 1×1 binning, and a Zeiss Fluar40×1.3 NA oil immersion objective. Fluorescence filters from Chroma wereas follows: GFP: HQ480/40, HQ535/50, Q505LP; Rhodamine: HQ545/30,HQ610/75, Q565LP. Images were background subtracted and contraststretched using Inovision ISEE software, then formatted for displayusing Adobe Photoshop software.

Results

To assess the ability of the peptide to maintain tight binding afterlabeling with dye, the affinity of the labeled peptide-peptide taginteraction was determined by equilibrium fluorescence titration,monitoring the changes in anisotropy and fluorescence intensity (seeFIG. 18). High affinity was critical for live cell fluorescence imagingto maximize the proportion of labeled peptide bound to the targetedprotein. Unattached labeled peptide could reduce sensitivity bygenerating uniform fluorescence background, and higher free peptideconcentrations could perturb normal cell function or generateappreciable nonspecific binding. Upon binding, the labeled peptideshowed a drop in quantum yield and a more than 230% increase inrhodamine anisotropy, indicating considerable reduction in rotation ofthe dye. Fitting the anisotropy values and peptide concentrations to aquadratic equation describing a simple 1:1 binding interaction (seeexperimental procedures) indicated that the interaction had a Kd valueof 5.4±1.1 nM. This high affinity suggested that there was little or noinfluence of the dye on the peptide-peptide interaction.

Next, α-actinin in living cells was visualized and the fluorescence ofthe GFP-fusion protein was compared to that of the peptide labeled withrhodamine. An α-actinin construct with GFP fused to the N-terminus andthe tag peptide on the C-terminus was expressed in Cos-7 cells. Thesecells were loaded with rhodamine peptide through microinjection. Theconcentration of the rhodamine-labeled peptide was optimized at 20 μM tominimize background fluorescence while clearly labeling the taggedpeptide. We had determined previously that a uniform fluorescencebackground is not a serious obstacle using other fluorescently labeledproteins, including rhodamine actin in cytoskeletal fibers, whichproduces a physiologically normal background of unpolymerized protein.

FIGS. 19 a and 19 c show GFP images and FIGS. 19 b and 19 d showrhodamine images taken of the same cells. The fluorescence from adifferent cell is shown in FIG. 20. The peptide tag gave remarkablydetailed images of α-actinin distribution, which was difficult todistinguish from those obtained using GFP. Controls in which only therhodamine-peptide or the α-actinin GFP were present in the cell provedthat the colocalization of the rhodamine and GFP signals was not duesimply to imperfect selectivity of the fluorescence filters used toseparate rhodamine and GFP signals. (See FIG. 21.) These controls alsodemonstrated that binding to other leucine zipper proteins or undesiredsites was not a problem, although a slight affinity of rhodamine for thenuclear envelope could be seen.

An important advantage of our labeling methodology is the ability tolabel proteins that cannot be readily isolated or reintroduced in thecell. This was demonstrated by fusing the tag peptide to the C-terminusof the F_(c)εRI, a membrane-spanning protein that remains in theendoplasmic reticulum unless co-expressed with the F_(c)εRI γ-subunit.The tagged protein was expressed in Cos-7 cells together with a GFPfusion of the ER-resident major histocompatability complex Iglycoprotein D_(b) (MHC), used here as an ER marker.

FIG. 22 shows that the ER-localized F_(c)εRI α-chain was clearlyvisualized using the rhodamine-tagged peptide. The distribution of thetwo proteins was not identical, with the Fc receptor showing someconcentrations in the perinuclear region. Controls using eachfluorescent species alone in cells showed that results were not due tobleedthrough of emission from one fluorophore into the image of theother, and that localizations were not the result of nonspecific binding(data not shown).

In summary, this approach provides access to many proteins which werepreviously very difficult to label with synthetic fluorophores for livecell experiments. Unlike other methods requiring relatively difficultsynthesis of reagents or bulky antibody tags, this procedure can beaccomplished using routine cloning and peptide synthesis procedures.

All publications, patents, and patent documents are incorporated byreference herein, as though individually incorporated by reference. Theinvention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications can be made while remainingwithin the spirit and scope of the invention.

1. A peptide conjugate of formula (IV):

wherein R⁶ is a peptide, polypeptide or antibody; X is a direct bond ora linking group; R⁷ is hydrogen, (C₁-C₆)alkyl, an amino protectinggroup, or a radical comprising one or more aminooxy groups; Z is alinking group, or Z is a direct single bond or a double bond between Nand D; and D is a functional molecule; provided that when D is apeptide, N and D are not linked through a —C═N—O—CH₂—C(═O)— linkage inthe backbone of the peptide conjugate.
 2. The peptide conjugate of claim1 wherein R⁶ is an antibody.
 3. A method of identifying an optimalposition for placement of a functional molecule on a peptide having apeptide backbone and a known activity, which comprises making a seriesof peptide conjugates, each peptide conjugate having the same amino acidsequence and the same functional molecule, wherein the functionalmolecule is linked at a different location along the backbone of everypeptide conjugate in the series, and observing which functional moleculelocation does not substantially interfere with the known activity of thepeptide.
 4. The method of claim 3 wherein each peptide of said series ofpeptide conjugates comprises the peptide conjugate of claim
 1. 5. Amethod of identifying an optimal position for placement of a functionalmolecule in a polypeptide having a known activity and an identifiedpeptide segment for attachment of the functional molecule, whichcomprises: a) making a series of peptide conjugates, each peptideconjugate having the amino acid sequence of the identified peptidesegment and the same functional molecule, wherein the functionalmolecule is linked at a different location along the backbone of everypeptide conjugate in the series; b) placing a peptide conjugate within,or at the end of, each polypeptide of a series of polypeptides to createa series of polypeptide conjugates each having the functional moleculeat a different location; and c) observing which functional moleculelocation does not substantially interfere with the known activity of thepolypeptide.
 6. The method of claim 5 wherein each peptide of saidseries of peptides comprises the peptide conjugate of claim
 1. 7. Themethod of claim 5 wherein each peptide conjugate is placed within, or atthe end of, each polypeptide by natural chemical ligation,intein-mediated protein ligation or chemical ligation.
 8. A method ofidentifying an optimal position for placement of anenvironmentally-sensitive functional molecule on a peptide biosensorhaving a backbone, which comprises making a series of peptideconjugates, each peptide conjugate having the same amino acid sequenceand the same functional molecule, wherein the functional molecule is ata different location along the backbone of every peptide conjugate inthe series, and observing which functional molecule location providesthe strongest signal change in response to an environmental change inthe peptide conjugate.
 9. The method of claim 8 wherein each peptideconjugate of said series of peptide conjugates comprises the peptideconjugate of claim
 1. 10. The method of claim 8 wherein each peptideconjugate is part of a polypeptide, antibody, antibody fragment,antibody fragment with linked chains or antibody with linked chains. 11.The method of claim 8 wherein said signal change is a change inphosphorescence, fluorescence emission intensity, fluorescence lifetime,fluorescence excitation wavelength or fluorescence emission wavelength.12. The method of claim 8 wherein said environmental change in saidpeptide conjugate is interaction with a target.
 13. The method of claim8 which further comprises observing the binding affinity of each peptideconjugate for target or the binding selectivity of each peptideconjugate for target.
 14. A method of identifying an optimal positionfor placement of an environmentally-sensitive functional molecule in apolypeptide having a known activity and an identified peptide segmentfor attachment of the functional molecule, which comprises: a) making aseries of peptide conjugates, each peptide conjugate having the aminoacid sequence of the identified peptide segment and the sameenvironmentally-sensitive functional molecule, wherein theenvironmentally-sensitive functional molecule is linked at a differentlocation along the backbone of every peptide conjugate in the series; b)placing a peptide conjugate within, or at the end of, each polypeptideof a series of polypeptides to create a series of polypeptide conjugateseach having the environmentally-sensitive functional molecule at adifferent location; and c) observing which functional molecule locationprovides the strongest signal change in response to an environmentalchange in the polypeptide conjugate.
 15. The method of claim 14 whereineach peptide conjugate of said series of peptide conjugates comprisesthe peptide conjugate of claim
 1. 16. The method of claim 14 whereineach peptide conjugate is placed within, or at the end of, eachpolypeptide by natural chemical ligation, intein-mediated proteinligation or chemical ligation.
 17. The method of claim 14 wherein saidsignal change is a change in phosphorescence, fluorescence emissionintensity, fluorescence lifetime, fluorescence excitation wavelength orfluorescence emission wavelength.
 18. The method of claim 14 whereinsaid environmental change in said protein conjugate is interaction witha target or a change in activation state of a target.
 19. The method ofclaim 14 which further comprises observing the binding affinity of eachpeptide conjugate for target or the binding selectivity of each peptideconjugate for target.
 20. A polypeptide biosensor which comprises thepeptide conjugate of claim
 1. 21. The polypeptide biosensor of claim 20which comprises a MAP a p21 activated kinase peptide capable of bindingRac, a Wiscott-Aldrich Syndrome Protein peptide capable of bindingcdc42, or a portion of a leucine zipper capable of binding anotherportion of a leucine zipper.
 22. A fusion protein comprising abiologically active Rho GTPase protein domain operatively linked to afluorescent protein, wherein said Rho GTPase protein domain is capableof binding GTP and forming an activated GTPase:GTP complex.
 23. Thefusion protein of claim 22 wherein said Rho GTPase protein domain isderived from a Rac, Rho or cdc42 protein.
 24. The fusion protein ofclaim 22 wherein said fluorescence protein is green fluorescenceprotein, cyan fluorescence protein, red fluorescence protein, yellowfluorescence protein, enhanced green fluorescence protein or enhancedyellow fluorescence protein.
 25. A nucleic acid encoding the fusionprotein of claim
 22. 26. An expression vector capable of expressing thefusion protein encoded by the nucleic acid of claim
 25. 27. A cellcomprising the expression vector of claim
 26. 28. A method for detectingGTP activation of a Rho GTPase protein in a live cell comprising: a)introducing a Rho GTPase protein to said cell, wherein said Rho GTPaseprotein is operatively linked to a fluorescence dye capable ofundergoing fluorescence resonance energy transfer; b) introducing apolypeptide biosensor into said cell, wherein said polypeptide biosensorcomprises a polypeptide capable of binding a GTP-activated Rho GTPaseprotein, and wherein said polypeptide is operatively linked to afluorescent dye which can undergo fluorescence resonance energy transferwith said fluorescence dye on said GTP-activated Rho GTPase protein whensaid polypeptide biosensor interacts with said GTP-activated Rho GTPase;and c) observing fluorescence emissions from said polypeptide biosensorwithin said live cell.
 29. The method of claim 28 which furthercomprises observing fluorescence emissions from said fluorescence dye onsaid GTP-activated Rho GTPase.
 30. The method of claim 28 wherein saidpolypeptide biosensor comprises the peptide conjugate of claim
 1. 31. Amethod for detecting GTP activation of a Rho GTPase protein, whichcomprises: a) contacting a polypeptide biosensor with a test substance,wherein said polypeptide biosensor comprises a polypeptide capable ofbinding a GTP-activated Rho GTPase protein, and wherein said polypeptideis operatively linked to an environmentally sensitive dye; and b)observing a signal from said polypeptide; wherein said environmentallysensitive dye will emit a signal of a different lifetime, intensity orwavelength when said polypeptide biosensor is bound to saidGTP-activated Rho GTPase protein than when said polypeptide biosensor isnot bound.
 32. The method of claim 31 wherein said polypeptide biosensorcomprises the peptide conjugate of claim
 1. 33. The method of claim 31wherein said polypeptide biosensor comprises the protein binding domainof p21 protein kinase or the protein binding domain of Wiscott Aldrichsyndrome protein.
 34. The method of claim 31 wherein said Rho GTPaseprotein domain is derived from a Rac, Rho or cdc42 protein.
 35. Themethod of claim 31 which further comprises quantifying the amount ofGTP-activated Rho GTPase protein.
 36. A method for detecting GTPactivation of a Rho GTPase protein in a live cell comprising: a)introducing a polypeptide biosensor into said cell, wherein saidpolypeptide biosensor comprises a polypeptide capable of binding aGTP-activated Rho GTPase protein, and wherein said polypeptide isoperatively linked to an environmentally sensitive dye; and b) observinga signal from said environmentally sensitive dye within said live cell;wherein said environmentally sensitive dye will emit a signal of adifferent lifetime, intensity or wavelength when said polypeptidebiosensor is bound to said GTP-activated Rho GTPase protein than whensaid polypeptide biosensor is not bound.
 37. The method of claim 36wherein said polypeptide biosensor comprises the peptide conjugate ofclaim
 1. 38. The method of claim 36 wherein said polypeptide biosensorcomprises the protein binding domain of p21 protein kinase or theprotein binding domain of Wiscott Aldrich syndrome protein.
 39. Themethod of claim 36 wherein said Rho GTPase protein domain is derivedfrom a Rac, Rho or cdc42 protein.
 40. The method of claim 36 whichfurther comprises quantifying the amount of GTP-activated Rho GTPaseprotein.
 41. A method for detecting binding of an antibody to an antigenwhich comprises reacting an antibody comprising the peptide conjugate ofclaim 1 with an antigen and detecting an antibody-antigen complex.
 42. Amethod for detecting binding of an antigen to an antibody whichcomprises reacting an antigen comprising the peptide conjugate of claimwith an antibody and detecting an antibody-antigen complex.
 43. Afluorescent compound of the formula:

wherein: each m is separately an integer ranging from 1-3; n is aninteger ranging from 0 to 5; R⁸, R¹¹ and R¹² are separately CO, SO₂,C═C(CN)₂, S, O or C(CH₃)₂; each R¹³ is alkyl, branched alkyl orheterocyclic ring derivatized with charged groups to enhance watersolubility and enhance photostability; each R⁹ and R¹⁰ is separately analkyl chain derivatized with charged groups to enhance water solubilityor with reactive groups for conjugation to other molecules.
 44. Thecompound of claim 43 wherein each R⁹ and R¹⁰ is separately sulfonate,amide or ether.
 45. The compound of claim 43 wherein each R⁹ and R¹⁰ isseparately —NH—(C═O)—CH₂-halide, amine, maleimide, —N═C═O, —N═C═S, acylhalide, succinimidyl ester, sulfosuccinimidyl ester, sulfonyl halide,sulfonyl azide, alcohol, thiol, semicarbazide, hydrazine orhydroxylamine.
 46. The compound of claim 43 wherein each R⁹ and R¹⁰ isseparately carboxylic acid, alkali or alkaline earth metal salt ofcarboxylic acid, carboxylic acid activated by carbodiimide, acylchloride, succinimidyl, sulfosuccinimidyl ester or COORx, wherein Rx isphenol or naphtol further substituted by at least one strong electronwithdrawing group.
 47. The compound of claim 43 having the formula:


48. The compound of claim 43 having the formula:


49. The compound of claim 43 having the formula:


50. A peptide biosensor comprising the compound of claim
 43. 51. Aprotein, polypeptide, peptide, antibody, antibody fragment or nucleicacid linked to the compound of claim
 43. 52. A method of detecting thelocation of a cellular protein within a living cell which comprises: a.providing the living cell with a biosensor capable of binding to a tagon the cellular protein; and b. detecting the location of a functionalmolecule on the biosensor within the living cell; wherein said tag is apeptide segment that has been fused to the cellular protein.
 53. Themethod of claim 52 wherein said tag has SEQ ID NO:16.
 54. The method ofclaim 52 wherein said functional molecule is the compound of claim 43.55. The method of claim 52 wherein said cellular protein is calmodulin,Rho GTPase, rac, cdc42, mitogen-activated protein kinase, Erk1, Erk2,Erk3, Erk4, IgE receptor (F_(c)εRI) actin, α-actinin, myosin, or a majorhistocompatibility protein.
 56. A method of detecting the location of acellular protein within a living cell which comprises: a. providing theliving cell with a biosensor capable of binding to a tag on the cellularprotein; and b. detecting the location of a functional molecule on thebiosensor within the living cell; wherein said tag is a peptide segmentthat has been fused to the cellular protein and said biosensor comprisesa functional molecule consisting of a fluorescent dye or a fluorescentprotein.
 57. The method of claim 56 wherein said tag has SEQ ID NO: 16.58. The method of claim 56 wherein said functional molecule is thecompound of claim
 43. 59. The method of claim 56 wherein said cellularprotein is calmodulin, Rho GTPase, rac, cdc42, mitogen-activated proteinkinase, Erk1, Erk2, Erk3, Erk4, IgE receptor (F_(c)εRI) actin,α-actinin, myosin, or a major histocompatibility protein.
 60. A methodof detecting the location of a cellular protein within a living cellwhich comprises: c. providing the living cell with a biosensor capableof binding to a tag on the cellular protein; and d. detecting thelocation of a functional molecule on the biosensor within the livingcell; wherein said tag is a peptide segment that has been fused to thecellular protein, and said functional molecule is a fluorescent compoundof claim
 43. 61. The method of claim 60 wherein said tag has SEQ IDNO:16.
 62. The method of claim 60 wherein said functional molecule isthe compound of claim
 43. 63. The method of claim 60 wherein saidcellular protein is calmodulin, Rho GTPase, rac, cdc42,mitogen-activated protein kinase, Erk1, Erk2, Erk3, Erk4, IgE receptor(F_(c)εRI) actin, α-actinin, myosin, or a major histocompatibilityprotein.
 64. A method of attaching a biosensor to a cellular proteinwithin a living cell which comprises, providing the living cell with abiosensor capable of binding to a tag on the cellular protein, whereinsaid tag is a peptide segment that has been fused to the cellularprotein expressed by the living cell.
 65. The method of claim 64 whereinsaid biosensor comprises the peptide-conjugate of claim
 1. 66. Themethod of claim 64 wherein said peptide-conjugate comprises afluorescent compound of claim
 43. 67. A nucleic acid encoding the tagfused to the cellular protein of claim
 64. 68. An isolated vectorcomprising the nucleic acid of claim
 67. 69. An expression vectorcapable of expressing the tag fused to the cellular protein of claim 64.70. A cell comprising the expression vector of claim 69.